{"data":[{"id":1,"pmid":25103205,"title":"Detection of Middle East respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (RT-LAMP)","year":2014,"journal":"Virology Journal","authors":"Kazuya Shirato, Takuya Yano, Syouhei Senba, Shigehiro Akachi, Takashi Kobayashi, Takamichi Nishinaka, Tsugunori Notomi, Shutoku Matsuyama","doi":"10.1186\/1743-422X-11-139","country":"Japan","institute":"National Institute of Infectious Disease","deparment":"Department of Virology III","abstract":"Background: The first documented case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) occurred in 2012, and outbreaks have continued ever since, mainly in Saudi Arabia. MERS-CoV is primarily diagnosed using a real-time RT-PCR assay, with at least two different genomic targets required for a positive diagnosis according to the case definition of The World Health Organization (WHO) as of 3 July 2013. Therefore, it is urgently necessary to develop as many specific genetic diagnostic methods as possible to allow stable diagnosis of MERS-CoV infections.\nMethods: Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is a genetic diagnostic method used widely for the detection of viral pathogens, which requires only a single temperature for amplification, and can be completed in less than 1 h. This study developed a novel RT-LAMP assay for detecting MERS-CoV using primer sets targeting a conserved nucleocapsid protein region.\nResults: The RT-LAMP assay was capable of detecting as few as 3.4 copies of MERS-CoV RNA, and was highly specific, with no cross-reaction to other respiratory viruses. Pilot experiments to detect MERS-CoV from medium containing pharyngeal swabs inoculated with pre-titrated viruses were also performed. The RT-LAMP assay exhibited sensitivity similar to that of MERS-CoV real-time RT-PCR.\nConclusions: These results suggest that the RT-LAMP assay described here is a useful tool for the diagnosis and epidemiologic surveillance of human MERS-CoV infections.","lamp_id":[{"id":"LPB00001","pathogen":"MERS-CoV","target":"N"}]},{"id":2,"pmid":28119682,"title":"One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV","year":2017,"journal":"Frontiers in Microbiology","authors":"Se Hee Lee, Yun Hee Baek, Yang-Hoon Kim, Young-Ki Choi, Min-Suk Song, Ji-Young Ahn","doi":"10.3389\/fmicb.2016.02166","country":"South Korea","institute":"Chungbuk National University","deparment":"School of Biological Sciences","abstract":"Due to the limitation of rapid development of specific antiviral drug or vaccine for novel emerging viruses, an accurate and rapid diagnosis is a key to manage the virus spread. We developed an efficient and rapid method with high specificity for the Middle East Respiratory Syndrome coronavirus (MERS-CoV), based on one-pot reverse transcription loop-mediated isothermal amplification (one-pot RT-LAMP). A set of six LAMP primers [F3, B3, FIP, BIP, LF (Loop-F), and LB (Loop-B)] were designed using the sequence of nucleocapsid (N) gene with optimized RT-LAMP enzyme conditions: 100 U M-MLV RTase and 4 U Bst polymerase, implying that the reaction was able to detect four infectious viral genome copies of MERS-CoV within a 60 min reaction time period. Significantly, EvaGreen dye has better signal read-out properties in one-pot RT-LAMP reaction and is more compatible with DNA polymerase than SYBR green I. Isothermally amplified specific N genes were further evaluated using field-deployable microchamber devices, leading to the specific identification of as few as 0.4 infectious viral genome copies, with no cross-reaction to the other acute respiratory disease viruses, including influenza type A (H1N1 and H3N2), type B, human coronavirus 229E, and human metapneumovirus. This sensitive, specific and feasible method provides a large-scale technical support in emergencies, and is also applied as a sample-to-detection module in Point of Care Testing devices.","lamp_id":[{"id":"LPB00002","pathogen":"MERS-CoV","target":"N"}]},{"id":3,"pmid":15131154,"title":"Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Method for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus","year":2004,"journal":"Journal of Clinical Microbiology","authors":"Thi Cam Thai Hong, Quynh Le Mai, Duc Vuong Cuong, Manmohan Parida, Harumi Minekawa, Tsugunori Notomi, Futoshi Hasebe, Kouichi Morita","doi":"10.1128\/JCM.42.5.1956-1961.2004","country":"Japan","institute":"Institute of Tropical Medicine","deparment":"Department of Virology","abstract":"The development and evaluation of a one-step single-tube accelerated real-time quantitative reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay is reported for rapid detection of the severe acute respiratory syndrome coronavirus (SARS-CoV) replicase gene. A total of 49 samples (15 throat washes, 13 throat swabs, and 21 combined throat and nasal swabs) collected from patients admitted to the Hanoi-French and Ninhbinh hospitals in Vietnam during the SARS epidemic were evaluated and compared to conventional RT-PCR. The RT-LAMP assay demonstrated 100-fold-greater sensitivity, with a detection limit of 0.01 PFU. The sensitivity and specificity of RT-LAMP assay for detecting viral RNA in clinical specimens with regard to RT-PCR were 100 and 87%, respectively. The specificity of the RT-LAMP assay was further validated by restriction analysis as well as nucleotide sequencing of the amplified product. The concentration of virus in most of the clinical samples was 0.1 PFU (0.1 to 10(2) PFU), as determined from the standard curve of SARS RT-LAMP and based on the time of positivity. The assay procedure is quite simple, wherein the amplification is carried out in a single tube under isothermal conditions at 63 degrees C, and the result can be obtained in less than 1 h (as early as 11 min). Thus, the RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of SARS-CoV in developing countries.","lamp_id":[{"id":"LPB00003","pathogen":"SARS-CoV","target":"Rep"}]},{"id":4,"pmid":32226589,"title":"A Simple and Multiplex Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of SARS-CoV","year":2019,"journal":"BioChip Journal","authors":"Jin Hwa Kim, Minhee Kang, Eunkyoung Park, Doo Ryeon Chung, Jiyeon Kim, Eung Soo Hwang","doi":"10.1007\/s13206-019-3404-3","country":"Republic of Korea","institute":"Smart Healthcare Research Institute\/Sungkyunkwan University","deparment":"Biomedical Engineering Research Center\/Department of Medical Device Management and Research","abstract":"The current diagnosis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) relies on laboratory-based tests since its clinical features are nonspecific, unlike other respiratory pathogens. Therefore, the development of a rapid and simple method for on-site detection of SARS-CoV is crucial for the identification and prevention of future SARS outbreaks. In this study, a simple colorimetric and multiplex loop-mediated isothermal amplification (LAMP) assay was developed to rapid screening of severe acute respiratory syndrome-associated coronavirus (SARS-CoV). It can be visually detected based on color change and monitored in real-time with fluorescent signals. The performance of this assay, based on six primers targeting open reading frame (ORF1b) and nucleocapsid (N) genes located in different regions of the SARS-CoV, was compared with real-time RT-PCR assay using various concentrations of target genes. The detection limit of the LAMP assay was comparable to that of real-time RT-PCR assay and therefore a few target RNA to 10^4-10^5 copies could be detected within a short period of time (20-25 min). In addition, we established a multiplex real-time LAMP assay to simultaneously detect two target regions within the SARS-CoV genome. Two target sequences were amplified by specific primers in the same reaction tube and revealed that it was able to detect down to 10^5 copies. The standard curve had a linear relationship with similar amplification efficiencies. The LAMP assay results in shorter \"sample-to-answer\" time than conventional PCR method. Therefore, it is suitable not only for diagnosis of clinical test, but also for surveillance of SARS virus in developing countries.","lamp_id":[{"id":"LPB00004","pathogen":"SARS-CoV","target":"ORF1b"},{"id":"LPB00135","pathogen":"SARS-CoV","target":"N"}]},{"id":5,"pmid":16192434,"title":"Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae","year":2005,"journal":"Journal of Medical Microbiology","authors":"Ryoichi Saito, Yoshiki Misawa, Kyoji Moriya, Kazuhiko Koike, Kimiko Ubukata, Noboru Okamura","doi":"10.1099\/jmm.0.46071-0","country":"Japan","institute":"University of Tokyo Hospital\/Tokyo Medical and Dental University","deparment":"Department of Infection Control and Prevention\/Department of Microbiology and Immunology","abstract":"A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Mycoplasma pneumoniae was developed and evaluated. The assay specifically amplified only M. pneumoniae sequences, and no cross-reactivity was observed for other Mycoplasma species or respiratory bacterial species. The detection limit for this assay was found to be 2 x 10(2) copies, corresponding to 2-20 colour changing units of M. pneumoniae in 1 h, as observed in a real-time turbidimeter and electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis as well as direct sequencing of the amplified product. The assay was applied to 95 nasopharyngeal swab samples collected from patients or from healthy individuals, and compared to a real-time PCR assay in-house. A concordance of 100% was observed between the two assays. The LAMP assay is easy to perform, shows a rapid reaction and is inexpensive. It may therefore be applied in the routine diagnosis of M. pneumoniae infection in the clinical laboratory.","lamp_id":[{"id":"LPB00005","pathogen":"Mycoplasma pneumoniae","target":"P1"}]},{"id":6,"pmid":32900935,"title":"SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification","year":2020,"journal":"Proceedings of the National Academy of Sciences of the United States of America","authors":"Brian A Rabe, Constance Cepko","doi":"10.1073\/pnas.2011221117","country":"USA","institute":"Blavatnik Institute","deparment":"Department of Genetics","abstract":"The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.","lamp_id":[{"id":"LPB00006","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00063","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":7,"pmid":32868442,"title":"Rapid isothermal amplification and portable detection system for SARS-CoV-2","year":2020,"journal":"Proceedings of the National Academy of Sciences of the United States of America","authors":"Anurup Ganguli, Ariana Mostafa, Jacob Berger, Mehmet Y Aydin, Fu Sun, Sarah A Stewart de Ramirez, Enrique Valera, Brian T Cunningham, William P King, Rashid Bashir","doi":"10.1073\/pnas.2014739117","country":"USA","institute":"University of Illinois at Urbana-Champaign","deparment":"Department of Bioengineering\/Nick Holonyak Jr. Micro and Nanotechnology Laboratory","abstract":"The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per \u03bcL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.","lamp_id":[{"id":"LPB00007","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00009","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00012","pathogen":"SARS-CoV-2","target":"ORF8"},{"id":"LPB00014","pathogen":"SARS-CoV-2","target":"N"}]},{"id":8,"pmid":15635005,"title":"Detection of human influenza A viruses by loop-mediated isothermal amplification","year":2005,"journal":"Journal of Clinical Microbiology","authors":"Leo L M Poon, Cynthia S W Leung, Kwok H Chan, Jack H C Lee, Kwok Y Yuen, Yi Guan, Joseph S M Peiris","doi":"10.1128\/JCM.43.1.427-430.2005","country":"Hong Kong","institute":"University of Hong Kong","deparment":"Department of Microbiology","abstract":"Here we describe the use of the loop-mediated isothermal amplification (LAMP) method to detect human influenza viruses (H1 to H3). Our results were correlated 100% with results deduced from routine clinical diagnostic tests. In addition, we also developed a LAMP assay specific for human beta-actin cDNA as a quality control test.","lamp_id":[{"id":"LPB00008","pathogen":"Influenza A virus (H1N1)","target":"M"}]},{"id":9,"pmid":17052763,"title":"Diagnosis of human respiratory syncytial virus infection using reverse transcription loop-mediated isothermal amplification","year":2007,"journal":"Journal of Virological Methods","authors":"Kazuya Shirato, Hidekazu Nishimura, Masayuki Saijo, Michiko Okamoto, Masahiro Noda, Masato Tashiro, Fumihiro Taguchi","doi":"10.1016\/j.jviromet.2006.09.014","country":"Japan","institute":"National Institute of Infectious Diseases","deparment":"Department of Virology III","abstract":"Human respiratory syncytial virus (RSV) is a major causative agent of lower respiratory tract infections in children and the elderly. A reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was developed assay to amplify the genome of RSV subgroups A and B, in order to improve current diagnostic methods for RSV infection. The primer sets for RT-LAMP were designed using highly conserved nucleotide sequences in the matrix protein region of subgroups A and B, and were specific for each subgroup. The RT-LAMP efficiency was compared to virus isolation and a commercially available enzyme immunoassay (EIA) for RSV detection (BD Directigen EZ RSV test), using nasopharyngeal aspirates from 59 children with respiratory tract infections. The RT-LAMP was specific for RSV and could not detect other respiratory pathogens. 61% (36\/59) of children were positive by RT-LAMP, 34% (20\/59) by viral isolation, and 56% (26\/46) by EZ RSV. Of 16 specimens that were negative by both antigen detection and virus isolation, 12.5% (2\/16) were RT-LAMP positive. These results suggest that the RT-LAMP is more sensitive than other methods used to detect RSV. The RT-LAMP assay developed in this study may be useful for diagnostic and epidemiological studies of RSV infection.","lamp_id":[{"id":"LPB00010","pathogen":"RSV A","target":"M"},{"id":"LPB00011","pathogen":"HRSV B","target":"M"}]},{"id":10,"pmid":34429488,"title":"Development and validation of LAMP primer sets for rapid identification of Aspergillus fumigatus carrying the cyp51A TR46 azole resistance gene","year":2021,"journal":"Scientific Reports","authors":"Plinio Trabasso, Tetsuhiro Matsuzawa, Teppei Arai, Daisuke Hagiwara, Yuzuru Mikami, Maria Luiza Moretti, Akira Watanabe","doi":"10.1038\/s41598-021-96651-7","country":"Brazil","institute":"University of Campinas","deparment":"School of Medical Sciences","abstract":"Infections due to triazole-resistant Aspergillus fumigatus are increasingly reported worldwide and are associated with treatment failure and mortality. The principal class of azole-resistant isolates is characterized by tandem repeats of 34 bp or 46 bp within the promoter region of the cyp51A gene. Loop-mediated isothermal amplification (LAMP) is a widely used nucleic acid amplification system that is fast and specific. Here we describe a LAMP assay method to detect the 46 bp tandem repeat insertion in the cyp51A gene promoter region based on novel LAMP primer sets. It also differentiated strains with TR46 tandem repeats from those with TR34 tandem repeats. These results showed this TR46-LAMP method is specific, rapid, and provides crucial insights to develop novel antifungal therapeutic strategies against severe fungal infections due to A. fumigatus with TR46 tandem repeats.","lamp_id":[{"id":"LPB00013","pathogen":"Aspergillus fumigatus","target":"cyp51A"}]},{"id":11,"pmid":33951060,"title":"Low saliva pH can yield false positives results in simple RT-LAMP-based SARS-CoV-2 diagnostic tests","year":2021,"journal":"PLoS One","authors":"Cristina Uribe-Alvarez, Quynh Lam, Don A Baldwin, Jonathan Chernoff","doi":"10.1371\/journal.pone.0250202","country":"USA","institute":"Fox Chase Cancer Center","deparment":"Cancer Signaling and Epigenetics Department","abstract":"Diagnosis of any infectious disease is vital for opportune treatment and to prevent dissemination. RT-qPCR tests for detection of SARS-CoV-2, the causative agent for COVID-19, are ideal in a hospital environment. However, mass testing requires cheaper and simpler tests, especially in settings that lack sophisticated machinery. The most common current diagnostic method is based on nasopharyngeal sample collection, RNA extraction, and RT-qPCR for amplification and detection of viral nucleic acids. Here, we show that samples obtained from nasopharyngeal swabs in VTM and in saliva can be used with or without RNA purification in an isothermal loop-mediated amplification (LAMP)-based assay, with 60-93% sensitivity for SARS-CoV-2 detection as compared to standard RT-qPCR tests. A series of simple modifications to standard RT-LAMP published methods to stabilize pH fluctuations due to salivary acidity resulted in a significant improvement in reliability, opening new avenues for efficient, low-cost testing of COVID-19 infection.","lamp_id":[{"id":"LPB00015","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00016","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00108","pathogen":"SARS-CoV-2","target":"N"}]},{"id":12,"pmid":32276051,"title":"Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)","year":2020,"journal":"The Journal of Molecular Diagnostics","authors":"Gun-Soo Park, Keunbon Ku, Seung-Hwa Baek, Seong-Jun Kim, Seung Il Kim, Bum-Tae Kim, Jin-Soo Maeng","doi":"10.1016\/j.jmoldx.2020.03.006","country":"Republic of Korea","institute":"Korea Research Institute of Chemical Technology\/Korea Food Research Institute","deparment":"Center for Convergent Research of Emerging Virus Infection\/Research Group of Food Processing","abstract":"The coronavirus disease 2019 (COVID-19) pandemic now has >2,000,000 confirmed cases worldwide. COVID-19 is currently diagnosed using quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. We developed and evaluated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. RT-LAMP assays reported in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay to enable higher throughput.","lamp_id":[{"id":"LPB00017","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"},{"id":"LPB00018","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"}]},{"id":13,"pmid":35660994,"title":"RT-LAMP assay combining multi-fluorescent probes for SARS-CoV-2 RNA detection and variant differentiation","year":2022,"journal":"Talanta","authors":"Jadera Talap, Minzhe Shen, Lushan Yu, Su Zeng, Sheng Cai","doi":"10.1016\/j.talanta.2022.123644","country":"China","institute":"Zhejiang Province Key Laboratory of Anti-Cancer Drug Research","deparment":"Institute of Drug Metabolism and Pharmaceutical Analysis","abstract":"Simple and accurate testing tools for SARS-CoV-2 viral RNA detection are essential for the prevention of the spread of the virus and timely governmental actions. Herein, we present a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the simultaneous detection of ORF1ab and N gene fragments of SARS-CoV-2 in one pot. Using two primer sets and two molecular beacon (MB) probes respectively labelled with different fluorophore, positive results were obtained with a limit of detection of 20 and 2 copies\/\u03bcL for ORF1ab and N gene fragments, respectively. Moreover, the RT-LAMP based assay was applied to detect single-site differences in S genes using two one-step displacement (OSD) probes targeting wild-type and mutant (P681R mutation was chosen as model) genes. Through that, the wild type strain and P681R mutant variant were well distinguished from each other, and a preliminary observation was also made on other mutations at this site such as P681H. The proposed method has high sensitivity for quantification and high specificity for mutation differentiation. In addition, it does not require accurate sophisticated thermal cycler instrumentation and can be used in clinical settings in resource-limited regions.","lamp_id":[{"id":"LPB00019","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00020","pathogen":"SARS-CoV-2","target":"N"}]},{"id":14,"pmid":32306853,"title":"Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2","year":2020,"journal":"Emerging Microbes & Infections","authors":"Yun Hee Baek, Jihye Um, Khristine Joy C Antigua, Ji-Hyun Park, Yeonjae Kim, Sol Oh, Young-Il Kim, Won-Suk Choi, Seong Gyu Kim, Ju Hwan Jeong, Bum Sik Chin, Halcyon Dawn G Nicolas, Ji-Young Ahn, Kyeong Seob Shin, Young Ki Choi, Jun-Sun Park, Min-Suk Song","doi":"10.1080\/22221751.2020.1756698","country":"Republic of Korea","institute":"Chungbuk National University College of Medicine and Medical Research Institute","deparment":"Department of Microbiology","abstract":"The previous outbreaks of SARS-CoV and MERS-CoV have led researchers to study the role of diagnostics in impediment of further spread and transmission. With the recent emergence of the novel SARS-CoV-2, the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Hence, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the specific detection of SARS-CoV-2. The primer sets for RT-LAMP assay were designed to target the nucleocapsid gene of the viral RNA, and displayed a detection limit of 10^2 RNA copies close to that of qRT-PCR. Notably, the assay has exhibited a rapid detection span of 30 min combined with the colorimetric visualization. This test can detect specifically viral RNAs of the SARS-CoV-2 with no cross-reactivity to related coronaviruses, such as HCoV-229E, HCoV-NL63, HCoV-OC43, and MERS-CoV as well as human infectious influenza viruses (type B, H1N1pdm, H3N2, H5N1, H5N6, H5N8, and H7N9), and other respiratory disease-causing viruses (RSVA, RSVB, ADV, PIV, MPV, and HRV). Furthermore, the developed RT-LAMP assay has been evaluated using specimens collected from COVID-19 patients that exhibited high agreement to the qRT-PCR. Our RT-LAMP assay is simple to perform, less expensive, time-efficient, and can be used in clinical laboratories for preliminary detection of SARS-CoV-2 in suspected patients. In addition to the high sensitivity and specificity, this isothermal amplification conjugated with a single-tube colorimetric detection method may contribute to the public health responses and disease control, especially in the areas with limited laboratory capacities.","lamp_id":[{"id":"LPB00021","pathogen":"SARS-CoV-2","target":"N"}]},{"id":15,"pmid":32276116,"title":"Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay","year":2020,"journal":"Clinical Microbiology and Infection","authors":"C Yan, J Cui, L Huang, B Du, L Chen, G Xue, S Li, W Zhang, L Zhao, Y Sun, H Yao, N Li, H Zhao, Y Feng, S Liu, Q Zhang, D Liu, J Yuan","doi":"10.1016\/j.cmi.2020.04.001","country":"China","institute":"Capital Institute of Paediatrics","deparment":"","abstract":"Objective: To evaluate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and compare it with RT-PCR.\n\nMethods: We designed primers specific to the orf1ab and S genes of SARS-CoV-2. Total viral RNA was extracted using the QIAamp Viral RNA Mini Kit. We optimized the RT-LAMP assay, and evaluated it for its sensitivity and specificity of detection using real-time turbidity monitoring and visual observation.\n\nResults: The primer sets orf1ab-4 and S-123 amplified the genes in the shortest times, the mean (\u00b1SD) times were 18 \u00b1 1.32 min and 20 \u00b1 1.80 min, respectively, and 63\u00b0C was the optimum reaction temperature. The sensitivities were 2 \u00d7 10^1 copies and 2 \u00d7 10^2 copies per reaction with primer sets orf1ab-4 and S-123, respectively. This assay showed no cross-reactivity with 60 other respiratory pathogens. To describe the availability of this method in clinical diagnosis, we collected 130 specimens from patients with clinically suspected SARS-CoV-2 infection. Among them, 58 were confirmed to be positive and 72 were negative by RT-LAMP. The sensitivity was 100% (95% CI 92.3%-100%), specificity 100% (95% CI 93.7%-100%). This assay detected SARS-CoV-2 in a mean (\u00b1SD) time of 26.28 \u00b1 4.48 min and the results can be identified with visual observation.\n\nConclusion: These results demonstrate that we developed a rapid, simple, specific and sensitive RT-LAMP assay for SARS-CoV-2 detection among clinical samples. It will be a powerful tool for SARS-CoV-2 identification, and for monitoring suspected patients, close contacts and high-risk groups.","lamp_id":[{"id":"LPB00022","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00023","pathogen":"SARS-CoV-2","target":"S"}]},{"id":16,"pmid":32941977,"title":"Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2","year":2020,"journal":"Journal of Virological Methods","authors":"Abu Naser Mohon, Lisa Oberding, Jana Hundt, Guido van Marle, Kanti Pabbaraju, Byron M Berenger, Luiz Lisboa, Thomas Griener, Markus Czub, Cody Doolan, Venice Servellita, Charles Y Chiu, Alexander L Greninger, Keith R Jerome, Dylan R Pillai","doi":"10.1016\/j.jviromet.2020.113972","country":"Canada","institute":"University of Calgary","deparment":"Department of Microbiology, Immunology, and Infectious Diseases","abstract":"A novel reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2 has been developed. The LAMP assay achieves a comparable limit of detection (25-50 copies per reaction) to commonly used RT-PCR protocols using clinical samples quantified by digital droplet PCR. Precision, cross-reactivity, inclusivity, and limit of detection studies were performed according to regulatory standards. Clinical validation of dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 98.48 % (95 % CI 91.84%-99.96%) and NPA 100.00 % (95 % CI 93.84%-100.00%) based on the E gene and N2 gene reference RT-PCR methods. The method has implications for development of point of care technology using isothermal amplification.","lamp_id":[{"id":"LPB00024","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00025","pathogen":"SARS-CoV-2","target":"RdRp"}]},{"id":17,"pmid":32547882,"title":"Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2","year":2020,"journal":"PeerJ","authors":"Yee Ling Lau, Ilyiana Ismail, Nur Izati Mustapa, Meng Yee Lai, Tuan Suhaila Tuan Soh, Afifah Hassan, Kalaiarasu M Peariasamy, Yee Leng Lee, Yoong Min Chong, I-Ching Sam, Pik Pin Goh","doi":"10.7717\/peerj.9278","country":"Malaysia","institute":"University of Malaya","deparment":"Department of Parasitology","abstract":"Background: Highly sensitive real-time reverse transcription polymerase chain reaction (RT-qPCR) methods have been developed for the detection of SARS-CoV-2. However, they are costly. Loop-mediated isothermal amplification (LAMP) assay has emerged as a novel alternative isothermal amplification method for the detection of nucleic acid.\n\nMethods: A rapid, sensitive and specific real-time reverse transcription LAMP (RT-LAMP) assay was developed for SARS-CoV-2 detection.\n\nResults: This assay detected one copy\/reaction of SARS-CoV-2 RNA in 30 min. Both the clinical sensitivity and specificity of this assay were 100%. The RT-LAMP showed comparable performance with RT-qPCR. Combining simplicity and cost-effectiveness, this assay is therefore recommended for use in resource resource-limited settings.","lamp_id":[{"id":"LPB00026","pathogen":"SARS-CoV-2","target":"N"}]},{"id":18,"pmid":32315390,"title":"Rapid detection of COVID-19 coronavirus using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform","year":2020,"journal":"Clinical Chemistry","authors":"Lin Yu, Shanshan Wu, Xiaowen Hao, Xue Dong, Lingling Mao, Vicent Pelechano, Wei-Hua Chen, Xiushan Yin","doi":"10.1093\/clinchem\/hvaa102","country":"China","institute":"Shenyang University of Chemical Technology","deparment":"Applied Biology Laboratory","abstract":null,"lamp_id":[{"id":"LPB00027","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":19,"pmid":32626666,"title":"Development and Validation of a Rapid, Single-Step Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) System Potentially to Be Used for Reliable and High-Throughput Screening of COVID-19","year":2020,"journal":"Frontiers in Cellular and Infection Microbiology","authors":"Minghua Jiang, Weihua Pan, Amir Arasthfer, Wenjie Fang, Liyan Ling, Hua Fang, Farnaz Daneshnia, Jian Yu, Wanqing Liao, Hao Pei, Xiaojing Li, Cornelia Lass-Fl\u00f6rl","doi":"10.3389\/fcimb.2020.00331","country":"China","institute":"The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University","deparment":"Department of Laboratory Medicine","abstract":"Objectives: Development and validation of a single-step and accurate reverse transcriptase loop-mediated isothermal amplification technique (RT-LAMP) for rapid identification of SARS-CoV-2 relative to commercial quantitative reverse transcriptase real-time PCR (qRT-PCR) assays to allow prompt initiation of proper medical care and containment of virus spread. Methods: Primers showing optimal in-silico features were subjected to analytical sensitivity and specificity to assess the limit of detection (LOD) and cross-reaction with closely- and distantly-related viral species, and clinically prominent bacterial and fungal species. In order to evaluate the clinical utility, our RT-LAMP was subjected to a large number of clinical samples, including 213 negative and 47 positive patients, relative to two commercial quantitative RT-PCR assays. Results: The analytical specificity and sensitivity of our assay was 100% and 500 copies\/ml when serial dilution was performed in both water and sputum. Subjecting our RT-LAMP assay to clinical samples showed a high degree of specificity (99.5%), sensitivity (91.4%), positive predictive value (97.7%), and negative predictive value (98.1%) when used relative to qRT-PCR. Our RT-LAMP assay was two times faster than qRT-PCR and is storable at room temperature. A suspected case that later became positive tested positive using both our RT-LAMP and the two qRT-PCR assays, which shows the capability of our assay for screening purposes. Conclusions: We present a rapid RT-LAMP assay that could extend the capacity of laboratories to process two times more clinical samples relative to qRT-PCR and potentially could be used for high-throughput screening purposes when demand is increasing at critical situations.","lamp_id":[{"id":"LPB00028","pathogen":"SARS-CoV-2","target":"N"}]},{"id":20,"pmid":31370782,"title":"Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform","year":2019,"journal":"BMC Infectious Diseases","authors":"Su Jeong Ahn, Yun Hee Baek, Khristine Kaith S Lloren, Won-Suk Choi, Ju Hwan Jeong, Khristine Joy C Antigua, Hyeok-Il Kwon, Su-Jin Park, Eun-Ha Kim, Young-Il Kim, Young-Jae Si, Seung Bok Hong, Kyeong Seob Shin, Sungkun Chun, Young Ki Choi, Min-Suk Song","doi":"10.1186\/s12879-019-4277-8","country":"Republic of Korea","institute":"College of Medicine and Medical Research Institute","deparment":"Department of Microbiology","abstract":"Background: In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological\/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans.\n\nMethods: We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses).\n\nResults: We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%).\n\nConclusions: Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.","lamp_id":[{"id":"LPB00029","pathogen":"Influenza B virus","target":"NA"},{"id":"LPB00030","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00031","pathogen":"Influenza A virus (H3N2)","target":"HA"},{"id":"LPB00032","pathogen":"Influenza A virus (H5)","target":"HA"},{"id":"LPB00033","pathogen":"Influenza A virus (H7N9)","target":"HA"}]},{"id":21,"pmid":21545810,"title":"Development of loop-mediated isothermal amplification assay for detection of human coronavirus-NL63","year":2011,"journal":"Journal of Virological Methods","authors":"Krzysztof Pyrc, Aleksandra Milewska, Jan Potempa","doi":"10.1016\/j.jviromet.2011.04.024","country":"Poland","institute":"Jagiellonian University","deparment":"Microbiology Department","abstract":"Human coronavirus NL63 was identified in 2004 in the Netherlands. Due to the high prevalence and world-wide distribution of this pathogen, it is essential to develop a sensitive and specific detection assay suitable for use in a routine diagnostic laboratory. Techniques based on PCR or real-time PCR are laborious and expensive. Detailed analysis of the HCoV-NL63 genome permitted the identification of a conserved nucleic acid sequential motif, which was sufficient for the design of a loop-mediated isothermal amplification (LAMP) assay. Evaluation of the method showed that the test is specific to HCoV-NL63 and that it does not cross-react with other respiratory viruses. The detection limit was found to be 1 copy of RNA template per reaction in cell culture supernatants and clinical specimens.","lamp_id":[{"id":"LPB00034","pathogen":"HCoV-NL63","target":"N"}]},{"id":22,"pmid":23827787,"title":"Multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of influenza A\/H1, A\/H3 and influenza B can provide a specimen-to-result diagnosis in 40 min with single genome copy sensitivity","year":2013,"journal":"Journal of Clinical Virology","authors":"James Mahony, Sylvia Chong, David Bulir, Alexandra Ruyter, Ken Mwawasi, Daniel Waltho","doi":"10.1016\/j.jcv.2013.06.006","country":"Canada","institute":"McMaster University\/St. Joseph's Healthcare Hamilton","deparment":"Department of Pathology and Molecular Medicine\/Regional Virology and Chlamydiology Laboratory","abstract":"Background: Rapid isothermal amplification methods have recently been introduced and they offer significant advantages over PCR.\n\nObjective: To develop a rapid and sensitive M-LAMP assay for the detection of influenza A (H1 and H3) and B that does not require RNA extraction.\n\nStudy design: We designed six primers targeting the matrix genes of influenza H1 and H3 and the NS1 gene of influenza B and developed a M-LAMP assay using a commercially available Master Mix and a real time fluorometer (Genie II, Optigene, UK) that displays real time amplification, time to positivity and amplicon annealing temperature (Tm). M-LAMP was evaluated against PCR by testing 202 nasopharyngeal (NP) specimens.\n\nResults: Optimized M-LAMP was rapid with a mean amplification time of 12 min (compared with 90-120 min for PCR), had an analytical sensitivity of 1 genome equivalent (ge), and could distinguish influenza A including subtypes A\/H1 and A\/H3 from influenza B by Tm. M-LAMP detected 26\/28 influenza A\/H1, 27\/27 influenza A\/H3 and 39\/39 influenza B specimens and had a combined sensitivity and specificity for detecting influenza (A and B) of 97.9% (92\/94) and 100% (108\/108), respectively. The rapid amplification time of LAMP coupled with a novel 10-min specimen preparation procedure consisting of vortexing and heating in M-Swab diluent (Copan Italia) provided a rapid result.\n\nConclusions: M-LAMP had excellent sensitivity and specificity for detecting influenza A and B in NP specimens and when used together with a rapid specimen processing method provided a specimen-to-result diagnosis in 30 min.","lamp_id":[{"id":"LPB00035","pathogen":"Influenza A virus (H3N2)","target":"M"},{"id":"LPB00036","pathogen":"Influenza A virus (H1)","target":"M"},{"id":"LPB00037","pathogen":"Influenza B virus","target":"NS1"}]},{"id":23,"pmid":20071551,"title":"Development of a reverse transcription-loop-mediated isothermal amplification assay for detection of pandemic (H1N1) 2009 virus as a novel molecular method for diagnosis of pandemic influenza in resource-limited settings","year":2010,"journal":"Journal of Clinical Microbiology","authors":"Toru Kubo, Masanobu Agoh, Le Q Mai, Kiyoyasu Fukushima, Hidekazu Nishimura, Akinori Yamaguchi, Manabu Hirano, Akira Yoshikawa, Futoshi Hasebe, Shigeru Kohno, Kouichi Morita","doi":"10.1128\/JCM.01481-09","country":"Japan","institute":"Institute of Tropical Medicine","deparment":"Center for International Collaborative Research\/Department of Virology","abstract":"This paper reports on the development of a one-step, real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the hemagglutinin (HA) gene for the rapid molecular-based detection of pandemic (H1N1) 2009 virus. The detection limit of the pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay was same as that of the currently used real-time reverse transcription-PCR method. The assay detected the pandemic (H1N1) 2009 virus HA gene in 136 RNA samples extracted from nasopharyngeal swab specimens from Japanese and Vietnamese patients. No cross-reactive amplification with the RNA of other seasonal influenza viruses was observed, and the detection of specific viral genome targets in clinical specimens was achieved in less than 40 min. The sensitivity and specificity of the pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay obtained in this study were 97.8% and 100%, respectively. Use of the (H1N1) 2009 virus HA-specific RT-LAMP assay will enable the faster and easier diagnosis of pandemic (H1N1) 2009 virus infection, especially in resource-limited situations in developing countries.","lamp_id":[{"id":"LPB00038","pathogen":"Influenza A virus (H1N1)","target":"HA"}]},{"id":24,"pmid":32719001,"title":"A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples","year":2020,"journal":"Science Translational Medicine","authors":"Viet Loan Dao Thi, Konrad Herbst, Kathleen Boerner, Matthias Meurer, Lukas Pm Kremer, Daniel Kirrmaier, Andrew Freistaedter, Dimitrios Papagiannidis, Carla Galmozzi, Megan L Stanifer, Steeve Boulant, Steffen Klein, Petr Chlanda, Dina Khalid, Isabel Barreto Miranda, Paul Schnitzler, Hans-Georg Kr\u00e4usslich, Michael Knop, Simon Anders","doi":"10.1126\/scitranslmed.abc7075","country":"Germany","institute":"Heidelberg University","deparment":"Department of Infectious Diseases","abstract":"The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.","lamp_id":[{"id":"LPB00039","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00040","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":25,"pmid":35324900,"title":"Profiling RT-LAMP tolerance of sequence variation for SARS-CoV-2 RNA detection","year":2022,"journal":"PLoS One","authors":"Esta Tamanaha, Yinhua Zhang, Nathan A. Tanner","doi":"10.1371\/journal.pone.0259610","country":"","institute":"","deparment":"","abstract":"The ongoing SARS-CoV-2 pandemic has necessitated a dramatic increase in our ability to conduct molecular diagnostic tests, as accurate detection of the virus is critical in preventing its spread. However, SARS-CoV-2 variants continue to emerge, with each new variant potentially affecting widely-used nucleic acid amplification diagnostic tests. RT-LAMP has been adopted as a quick, inexpensive diagnostic alternative to RT-qPCR, but as a newer method, has not been studied as thoroughly. Here we interrogate the effect of SARS-CoV-2 sequence mutations on RT-LAMP amplification, creating 523 single point mutation \u201cvariants\u201d covering every position of the LAMP primers in 3 SARS-CoV-2 assays and analyzing their effects with over 4,500 RT-LAMP reactions. Remarkably, we observed only minimal effects on amplification speed and no effect on detection sensitivity at positions equivalent to those that significantly impact RT-qPCR assays. We also created primer sets targeting a specific short deletion and observed that LAMP is able to amplify even with a primer containing multiple consecutive mismatched bases, albeit with reduced speed and sensitivity. This highlights RT-LAMP as a robust technique for viral RNA detection that can tolerate most mutations in the primer regions. Additionally, where variant discrimination is desired, we describe the use of molecular beacons to sensitively distinguish and identify variant RNA sequences carrying short deletions. Together these data add to the growing body of knowledge on the utility of RT-LAMP and increase its potential to further our ability to conduct molecular diagnostic tests outside of the traditional clinical laboratory environment.","lamp_id":[{"id":"LPB00041","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":26,"pmid":32635743,"title":"Enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride","year":2020,"journal":"Biotechniques","authors":"Yinhua Zhang, Guoping Ren, Jackson Buss, Andrew J Barry, Gregory C Patton, Nathan A Tanner","doi":"10.2144\/btn-2020-0078","country":"USA","institute":"New England Biolabs","deparment":"","abstract":"Loop-mediated isothermal amplification (LAMP) is a versatile technique for detection of target DNA and RNA, enabling rapid molecular diagnostic assays with minimal equipment. The global SARS-CoV-2 pandemic has presented an urgent need for new and better diagnostic methods, with colorimetric LAMP utilized in numerous studies for SARS-CoV-2 detection. However, the sensitivity of colorimetric LAMP in early reports has been below that of the standard RT-qPCR tests, and we sought to improve performance. Here we report the use of guanidine hydrochloride and combined primer sets to increase speed and sensitivity in colorimetric LAMP, bringing this simple method up to the standards of sophisticated techniques and enabling accurate, high-throughput diagnostics.","lamp_id":[{"id":"LPB00042","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00043","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00044","pathogen":"SARS-CoV-2","target":"E"}]},{"id":27,"pmid":32822689,"title":"iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2","year":2020,"journal":"Virus Research","authors":"Zahir Ali, Rashid Aman, Ahmed Mahas, Gundra Sivakrishna Rao, Muhammad Tehseen, Tin Marsic, Rahul Salunke, Amit K. Subudhi, Sharif M. Hala, Samir M. Hamdan, Arnab Pain, Fadwa S. Alofi, Afrah Alsomali, Anwar M. Hashem, Asim Khogeer, Naif A.M. Almontashiri, Malak Abedalthagafi, Norhan Hassan, Magdy M. Mahfouz","doi":"10.1016\/j.virusres.2020.198129","country":"Saudi Arabia","institute":"King Abdullah University of Science and Technology","deparment":"Division of Biological Sciences","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.","lamp_id":[{"id":"LPB00045","pathogen":"SARS-CoV-2","target":"N"}]},{"id":28,"pmid":25467501,"title":"Combination of multiplex reverse-transcription loop-mediated isothermal amplification with an immunochromatographic strip for subtyping influenza A virus","year":2015,"journal":"Analytica Chimica Acta","authors":"Jae Hwan Jung, Seung Jun Oh, Yong Tae Kim, So Yeon Kim, Won-Jung Kim, Jaean Jung, Tae Seok Seo","doi":"10.1016\/j.aca.2014.10.020","country":"Republic of Korea","institute":"Korea Advanced Institute of Science and Technology (KAIST)","deparment":"Department of Chemical and Biomolecular Engineering (BK21+ Program) and Institute for the BioCentury","abstract":"Considering the fatal human victims and economic loss by the annual epidemic influenza virus, the development of a rapid and convenient genetic analysis methodology is demanding for timely on-site pathogen detection. In this study, we utilized reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for multiplex target gene amplification, and the resultant amplicons were analyzed on the immunochromatographic strip (ICS) for subtyping influenza A virus. Through the optimized primer design, reaction temperature and time, and concentration of enzymes (Bst DNA polymerase and AMV reverse transcriptase) and dNTP, the HA (H1, H3, and H5 gene) and conserved M gene were amplified. The ICS contains two test lines in addition to a control line in order to detect the presence of the HA and M gene, thereby informing us of influenza virus A type as well as its subtype (H1N1, H3N2, and H5N1). The combination of the multiplex RT-LAMP with the ICS could be complete in 40 min and the pathotyping and subtyping of influenza A virus were performed even with 10 copies of viral RNA templates. Moreover, the subtyping of clinical samples, which were obtained from patients infected by influenza A virus was successfully confirmed using the multiplex RT-LAMP and ICS techniques, showing great feasibility of our methodology for real sample analysis with high speed, simplicity and sensitivity.","lamp_id":[{"id":"LPB00046","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00047","pathogen":"Influenza A virus (H3N2)","target":"HA"},{"id":"LPB00048","pathogen":"Influenza A virus (H5N1)","target":"HA"},{"id":"LPB00049","pathogen":"Influenza A virus (H1N1)","target":"M"}]},{"id":29,"pmid":28435249,"title":"Detection of influenza viruses by coupling multiplex reverse-transcription loop-mediated isothermal amplification with cascade invasive reaction using nanoparticles as a sensor","year":2017,"journal":"International Journal of Nanomedicine","authors":"Yiyue Ge, Qiang Zhou, Kangchen Zhao, Ying Chi, Bin Liu, Xiaoyan Min, Zhiyang Shi, Bingjie Zou, Lunbiao Cui","doi":"10.2147\/IJN.S132670","country":"China","institute":"Jiangsu Provincial Center for Disease Control and Prevention","deparment":"Institute of Pathogenic Microbiology","abstract":"Influenza virus infections represent a worldwide public health and economic problem due to the significant morbidity and mortality caused by seasonal epidemics and pandemics. Sensitive and convenient methodologies for detection of influenza viruses are essential for further disease control. Loop-mediated isothermal amplification (LAMP) is the most commonly used method of nucleic acid isothermal amplification. However, with regard to multiplex LAMP, differentiating the ladder-like LAMP products derived from multiple targets is still challenging today. The requirement of specialized instruments has further hindered the on-site application of multiplex LAMP. We have developed an integrated assay coupling multiplex reverse transcription LAMP with cascade invasive reaction using nanoparticles (mRT-LAMP-CIRN) as a sensor for the detection of three subtypes of influenza viruses: A\/H1N1pdm09, A\/H3 and influenza B. The analytic sensitivities of the mRT-LAMP-CIRN assay were 10^1\u00a0copies of RNA for both A\/H1N1pdm09 and A\/H3, and 10^2\u00a0copies of RNA for influenza B. This assay demonstrated highly specific detection of target viruses and could differentiate them from other genetically or clinically related viruses. Clinical specimen analysis showed the mRT-LAMP-CIRN assay had an overall sensitivity and specificity of 98.3% and 100%, respectively. In summary, the mRT-LAMP-CIRN assay is highly sensitive and specific, and can be used as a cost-saving and instrument-free method for the detection of influenza viruses, especially for on-site use.","lamp_id":[{"id":"LPB00050","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00051","pathogen":"Influenza A virus (H3N2)","target":"M"},{"id":"LPB00052","pathogen":"Influenza B virus","target":"NS1"}]},{"id":30,"pmid":24925133,"title":"New method for the visual detection of human respiratory syncytial virus using reverse transcription loop-mediated amplification","year":2014,"journal":"Journal of Virological Methods","authors":"Yonglin Mu, Jiawei Zeng, Qianqian Chen, Jia Liu, Lili Wang, Fujia Yao, Meng Cui, Zhixiang He, Chiyu Zhang, Ming Xiao, Ke Lan","doi":"10.1016\/j.jviromet.2014.06.005","country":"China","institute":"Shanghai Normal University\/Institut Pasteur of Shanghai","deparment":"College of Life and Environmental Science\/Pathogen Diagnostic Center","abstract":"Human respiratory syncytial virus (HRSV) is a seasonal respiratory pathogen that causes respiratory infection in children and the elderly. A new, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid (within 1h), simultaneous detection of A and B group HRSV. Primers specific for groups A and B were designed to amplify the N and L genes of HRSV, respectively. A fluorescent dye, calcein, was used as an indicator for the endpoint visual detection and\/or real-time amplification of HRSV RNA. The detection limit of the new method was 281.17 50% tissue culture infective doses (TCID50)\/ml for HRSV A and 1.58 TCID50\/ml for HRSV B. To evaluate the validity of this method, a comparison with RT-PCR was performed using 77 nasopharyngeal swabs as samples. Both RT-LAMP and RT-PCR detected HRSV in 38 HRSV samples, yielding a positive rate of 49%. Of the RT-LAMP positive samples, 36 (95%) were also positive by RT-PCR, while two were negative by RT-PCR. Among the 36 RT-LAMP and RT-PCR positive samples, 11 belonged to HRSV group A, while 25 belonged to group B. The results show that the new RT-LAMP is simple, rapid and well suited for HRSV diagnosis, especially in a limited-resource setting.","lamp_id":[{"id":"LPB00053","pathogen":"HRSV A","target":"N"},{"id":"LPB00054","pathogen":"HRSV B","target":"L"}]},{"id":31,"pmid":33954120,"title":"Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid and On-Site Detection of Avian Influenza Virus","year":2021,"journal":"Frontiers in Cellular and Infection Microbiology","authors":"Mohsen Golabi, Marion Flodrops, Beatrice Grasland, Aaydha C. Vinayaka, Than Linh Quyen, Trieu Nguyen, Dang Duong Bang, Anders Wolff","doi":"10.3389\/fcimb.2021.652048","country":"Denmark","institute":"Technical University of Denmark","deparment":"Department of Biotechnology and Biomedicine","abstract":"Avian influenza virus (AIV) outbreaks occur frequently worldwide, causing a potential public health risk and great economic losses to poultry industries. Considering the high mutation rate and frequent genetic reassortment between segments in the genome of AIVs, emerging new strains are a real threat that may infect and spread through the human population, causing a pandemic. Therefore, rapid AIV diagnostic tests are essential tools for surveillance and assessing virus spreading. Real-time reverse transcription PCR (rRT-PCR), targeting the matrix gene, is the main official standard test for AIV detection, but the method requires well-equipped laboratories. Reverse transcription Loop-Mediated Isothermal Amplification (RT-LAMP) has been reported as a rapid method and an alternative to PCR in pathogen detection. The high mutation rate in the AIV genome increases the risk of false negative in nucleic acid amplification methods for detection, such as PCR and LAMP, due to possible mismatched priming. In this study, we analyzed 800 matrix gene sequences of newly isolated AIV in the EU and designed a highly efficient LAMP primer set that covers all AIV subtypes. The designed LAMP primer set was optimized in real-time RT-LAMP (rRT-LAMP) assay. The rRT-LAMP assay detected AIV samples belonging to nine various subtypes with the specificity and sensitivity comparable to the official standard rRT-PCR assay. Further, a two-color visual detection RT-LAMP assay protocol was adapted with the aim to develop on-site diagnostic tests. The on-site testing successfully detected spiked AIV in birds oropharyngeal and cloacal swabs samples at a concentration as low as 100.8 EID50 per reaction within 30 minutes including sample preparation. The results revealed a potential of this newly developed rRT-LAMP assay to detect AIV in complex samples using a simple heat treatment step without the need for RNA extraction.","lamp_id":[{"id":"LPB00055","pathogen":"Influenza A virus (H1N1)","target":"M"}]},{"id":32,"pmid":29896174,"title":"A Rapid and Specific Assay for the Detection of MERS-CoV","year":2018,"journal":"Frontiers in Microbiology","authors":"Pei Huang , Hualei Wang, Zengguo Cao, Hongli Jin, Hang Chi, Jincun Zhao, Beibei Yu, Feihu Yan, Xingxing Hu, Fangfang Wu, Cuicui Jiao, Pengfei Hou, Shengnan Xu, Yongkun Zhao, Na Feng, Jianzhong Wang, Weiyang Sun, Tiecheng Wang, Yuwei Gao, Songtao Yang, Xianzhu Xia","doi":"10.3389\/fmicb.2018.01101","country":"China","institute":"Jilin Agricultural University\/Institute of Military Veterinary","deparment":"Animal Science and Technology College\/Key Laboratory of Jilin Province for Zoonosis Prevention and Control","abstract":"Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel human coronavirus that can cause human respiratory disease. The development of a detection method for this virus that can lead to rapid and accurate diagnosis would be significant. In this study, we established a nucleic acid visualization technique that combines the reverse transcription loop-mediated isothermal amplification technique and a vertical flow visualization strip (RT-LAMP-VF) to detect the N gene of MERS-CoV. The RT-LAMP-VF assay was performed in a constant temperature water bath for 30 min, and the result was visible by the naked eye within 5 min. The RT-LAMP-VF assay was capable of detecting 2 \u00d7 10^1 copies\/\u03bcl of synthesized RNA transcript and 1 \u00d7 10^1 copies\/\u03bcl of MERS-CoV RNA. The method exhibits no cross-reactivities with multiple CoVs including SARS-related (SARSr)-CoV, HKU4, HKU1, OC43 and 229E, and thus exhibits high specificity. Compared to the real-time RT-PCR (rRT-PCR) method recommended by the World Health Organization (WHO), the RT-LAMP-VF assay is easy to handle, does not require expensive equipment and can rapidly complete detection within 35 min.","lamp_id":[{"id":"LPB00056","pathogen":"MERS-CoV","target":"N"}]},{"id":33,"pmid":29253497,"title":"Evaluation of clinical applicability of reverse transcription-loop-mediated isothermal amplification assay for detection and subtyping of Influenza A viruses","year":2018,"journal":"Journal of Virological Methods","authors":"Vikrant Sharma, Dhruva Chaudhry, Samander Kaushik","doi":"10.1016\/j.jviromet.2017.12.005","country":"India","institute":"Maharshi Dayanand University","deparment":"Centre for Biotechnology","abstract":"Background: Influenza A viruses (IAVs) have always remain a serious concern for the global economy and public health. A rapid, specific and sensitive detection method is always needed to control the influenza in its early stages by timely intervention of therapy and early clinical management.\n\nObjectives: To develop RT-LAMP assays for detection of influenza A viruses, their further subtyping into seasonal (H1N1, H3N2) and novel pandemic H1N1 viruses and to evaluate clinical applicability of optimized RT-LAMP assays on patients' samples.\n\nStudy design: In this study, we optimized RT-LAMP assay to detect IAVs by using primers against matrix gene and subtyping of IAVs was done by using primers against hemagglutinin gene. Optimized RT-LAMP assays were applied on clinical samples from patients having influenza like illness and results were compared with conventional one-step RT-PCR and real-time RT-PCR.\n\nResults: RT-LAMP assays successfully detected and differentiated IAVs into H1N1, H3N2 and pdm09\/H1N1 subtypes. One hundred and sixty seven clinical swab samples from influenza suspected patients were taken and tested with RT-LAMP assay, detecting 30 (17.9%) samples positive for Influenza A virus. Out of 30 samples, 21, 7 and 2 were found positive for pdm09\/H1N1, H3N2 and seasonal H1 respectively. Conventional one-step RT-PCR detected a total of 27 (16.2%) samples for influenza A and further subtyping showed 20 and 7 samples positive for pdm09\/H1N1 and H3N2 virus respectively whereas none was found positive for seasonal H1N1. RT-LAMP assay demonstrated higher sensitivity (93.8%) than conventional RT-PCR (84.4%) for influenza A viruses detection in clinical samples.\n\nConclusions: RT-LAMP assay is rapid, sensitive, specific and cost effective method for detection of influenza A viruses than conventional one-step RT-PCR and it can serve as a good alternate for diagnosis and surveillance studies during influenza outbreaks in resource-limited setups of developing countries.","lamp_id":[{"id":"LPB00057","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00058","pathogen":"Influenza A virus (H3N2)","target":"HA"},{"id":"LPB00149","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00198","pathogen":"Influenza A virus (H1N1)","target":"M"}]},{"id":34,"pmid":21328369,"title":"Mobile and accurate detection system for infection by the 2009 pandemic influenza A (H1N1) virus with a pocket-warmer reverse-transcriptase loop-mediated isothermal amplification","year":2011,"journal":"Journal of Medical Virology","authors":"Ben Hatano, Megumi Goto, Hitomi Fukumoto, Takeyuki Obara, Takayuki Maki, Go Suzuki, Tetsuo Yamamoto, Kohsuke Hagisawa, Yoshitaro Matsushita, Tatsuya Fujii, Toshihiko Imakiire, Yuichi Kikuchi, Ryota Takahashi, Mie Kanai, Kaku Tamura, Tomoko Izumi, Yukihiro Takahashi, Yujiro Iwamoto, Satoshi Mimura, Yasuo Mukai, Kazue Takita, Hiroki Takeo, Ryuichi Kitamura, Eiichi Shimizu, Koji Fukushima, Yukiya Hakozaki, Akimi Uehata, Masao Sakai, Satoshi Ohshima, Toshiki Shirotani, Kunihiro Oba, Hideki Hasegawa, Tetsutaro Sata, Harutaka Katano","doi":"10.1002\/jmv.22031","country":"Japan","institute":"National Institute of Infectious Diseases","deparment":"Department of Pathology","abstract":"The 2009 pandemic H1N1 influenza A virus spread quickly worldwide in 2009. Since most of the fatal cases were reported in developing countries, rapid and accurate diagnosis methods that are usable in poorly equipped laboratories are necessary. In this study, a mobile detection system for the 2009 H1N1 influenza A virus was developed using a reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) kit with a disposable pocket-warmer as a heating device (designated as pwRT-LAMP). The pwRT-LAMP can detect as few as 100 copies of the virus--which is nearly as sensitive as real-time reverse-transcription polymerase chain reaction (RT-PCR) and does not cross-react with RNA of seasonal influenza viruses. To evaluate the usefulness of the pwRT-LAMP system, nasal swab samples were collected from 56 patients with flu-like symptoms and were tested. Real-time RT-PCR confirmed that the 2009 H1N1 influenza A virus was present in 27 of the 56 samples. Of these 27 positive samples, QuickVue Influenza A+B immunochromatography detected the virus in only 11 samples (11\/27; 40.7%), whereas the pwRT-LAMP system detected the virus in 26 of the 56 samples (26\/27 of the positive samples; 96.3%). These findings indicate that the mobile pwRT-LAMP system is an accurate diagnostic system for the 2009 H1N1 influenza A virus, and has great potential utility in diagnosing future influenza pandemics.","lamp_id":[{"id":"LPB00059","pathogen":"Influenza A virus (H1N1)","target":"HA"}]},{"id":35,"pmid":34878775,"title":"Detection of SARS-CoV-2 Virus Amplification Using a Crumpled Graphene Field-Effect Transistor Biosensor","year":2021,"journal":"ACS Sensors","authors":"Insu Park, Jongwon Lim, Seungyong You, Michael Taeyoung Hwang, Jaehong Kwon, Katherine Koprowski, Sungdae Kim, John Heredia, Sarah A. Stewart de Ramirez, Enrique Valera, Rashid Bashir","doi":"10.1021\/acssensors.1c01937","country":"","institute":"","deparment":"","abstract":"The rapid and unexpected spread of SARS-CoV-2 worldwide has caused unprecedented disruption to daily life and has brought forward critical challenges for public health. The disease was the largest cause of death in the United States in early 2021. Likewise, the COVID-19 pandemic has highlighted the need for rapid and accurate diagnoses at scales larger than ever before. To improve the availability of current gold standard diagnostic testing methods, the development of point-of-care devices that can maintain gold standard sensitivity while reducing the cost and providing portability is much needed. In this work, we combine the amplification capabilities of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) techniques with high-sensitivity end-point detection of crumpled graphene field-effect transistors (cgFETs) to develop a portable detection cell. This electrical detection method takes advantage of the ability of graphene to adsorb single-stranded DNA due to noncovalent \u03c0-\u03c0 bonds but not double-stranded DNA. These devices have demonstrated the ability to detect the presence of the SARS-CoV-2 virus in a range from 10 to 10^4 copies\/\u03bcL in 20 viral transport medium (VTM) clinical samples. As a result, we achieved 100% PPV, NPV, sensitivity, and specificity with 10 positive and 10 negative VTM clinical samples. Further, the cgFET devices can differentiate between positive and negative VTM clinical samples in 35 min based on the Dirac point shift. Likewise, the improved sensing capabilities of the crumpled gFET were compared with those of the traditional flat gFET devices.","lamp_id":[{"id":"LPB00060","pathogen":"SARS-CoV-2","target":"N"}]},{"id":36,"pmid":32088701,"title":"A quite sensitive fluorescent loop-mediated isothermal amplification for rapid detection of respiratory syncytial virus","year":2019,"journal":"Journal of Infection in Developing Countries","authors":"Yihong Hu, Zhenzhou Wan, Yonglin Mu, Yi Zhou, Jia Liu, Ke Lan, Chiyu Zhang","doi":"10.3855\/jidc.11549","country":"China","institute":"Institut Pasteur of Shanghai","deparment":"Pathogen Discovery and Big Data Center","abstract":"Introduction: Human respiratory syncytial virus (hRSV) is a common respiratory virus closely related to respiratory tract infection (RTI). Rapid and accurate detection of hRSV is urgently needed to reduce the high morbidity and mortality due to hRSV infection.\n\nMethodology: Here, we established a highly sensitive and specific reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of A and B group hRSV simultaneously. The specific primer sets for hRSV A and B groups were designed in the M and M2-2 gene, respectively. SYTO 9 was used as the fluorescent dye for real-time monitoring of the amplification of hRSV RNA without cross reaction between hRSV A and B.\n\nResults: The limit of detection (LOD) of our new method was 281.17 50% tissue culture infective doses (TCID50)\/mL for hRSV A and 1.58 TCID50\/mL for hRSV B. Using 90 clinical samples, a comparison to traditional RT-PCR was performed to validate this assay. The positivity rate of RT-LAMP and RT-PCR were 67.8% and 55.6%, respectively, and the positivity rate of RT-LAMP was significantly higher than RT-PCR (\u03c72 test, P < 0.01).\n\nConclusions: Compared with traditional RT-PCR method, the newly developed fluorescent RT-LAMP combined with well-designed primers and SYTO 9 is quite sensitive, specific, rapid and well applicable to hRSV clinical diagnosis.","lamp_id":[{"id":"LPB00061","pathogen":"HRSV A","target":"M"},{"id":"LPB00062","pathogen":"HRSV B","target":"M2-2"}]},{"id":37,"pmid":26377809,"title":"Reverse-transcription, loop-mediated isothermal amplification assay for the sensitive and rapid detection of H10 subtype avian influenza viruses","year":2015,"journal":"Virology Journal","authors":"Sisi Luo, Zhixun Xie, Liji Xie, Jiabo Liu, Zhiqin Xie, Xianwen Deng, Li Huang, Jiaoling Huang, Tingting Zeng, Mazhar I Khan","doi":"10.1186\/s12985-015-0378-1","country":"People's Republic of China","institute":"Guangxi Veterinary Research Institute","deparment":"Guangxi Key Laboratory of Animal Vaccines and New Technology","abstract":"Background: The H10 subtype avian influenza viruses (H10N4, H10N5 and H10N7) have been reported to cause disease in mammals, and the first human case of H10N8 subtype avian influenza virus was reported in 2013. Recently, H10 subtype avian influenza viruses (AIVs) have been followed more closely, but routine diagnostic tests are tedious, less sensitive and time consuming, rapid molecular detection assays for H10 AIVs are not available.\n\nMethods: Based on conserved sequences within the HA gene of the H10 subtype AIVs, specific primer sets of H10 subtype of AIVs were designed and assay reaction conditions were optimized. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was established for the rapid detection of H10 subtype AIVs. The specificity was validated using multiple subtypes of AIVs and other avian respiratory pathogens, and the limit of detection (LOD) was tested using concentration gradient of in vitro-transcribed RNA.\n\nResults: The established assay was performed in a water bath at 63 \u00b0C for 40 min, and the amplification result was visualized directly as well as under daylight reflections. The H10-RT-LAMP assay can specifically amplify H10 subtype AIVs and has no cross-reactivity with other subtypes AIVs or avian pathogens. The LOD of the H10-RT-LAMP assay was 10 copies per \u03bcL of in vitro-transcribed RNA.\n\nConclusions: The RT-LAMP method reported here is demonstrated to be a potentially valuable means for the detection of H10 subtype AIV and rapid clinical diagnosis, being fast, simple, and low in cost. Consequently, it will be a very useful screening assay for the surveillance of H10 subtype AIVs in underequipped laboratories as well as in field conditions.","lamp_id":[{"id":"LPB00064","pathogen":"Influenza A virus (H10N8)","target":"HA"}]},{"id":38,"pmid":32530929,"title":"Rapid detection of novel coronavirus\/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by reverse transcription-loop-mediated isothermal amplification","year":2020,"journal":"PLoS One","authors":"Laura E Lamb, Sarah N Bartolon, Elijah Ward, Michael B Chancellor","doi":"10.1371\/journal.pone.0234682","country":"USA","institute":"Beaumont Health System\/Oakland University William Beaumont School of Medicine","deparment":"Department of Urology","abstract":"Novel Corona virus\/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or 2019-nCoV), and the subsequent disease caused by the virus (coronavirus disease 2019 or COVID-19), is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for SARS-CoV-2 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in 30-45 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the SARS-CoV-2 nucleic sequence. RNA isolated from nasopharyngeal swabs collected from actual COVID-19 patients was also tested. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected SARS-CoV-2 in both simulated patient samples and clinical specimens. This test was performed in 30-45 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.","lamp_id":[{"id":"LPB00065","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"}]},{"id":39,"pmid":23761156,"title":"Development of a sensitive loop-mediated isothermal amplification assay that provides specimen-to-result diagnosis of respiratory syncytial virus infection in 30 minutes","year":2013,"journal":"Journal of Clinical Microbiology","authors":"James Mahony, Sylvia Chong, David Bulir, Alexandra Ruyter, Ken Mwawasi, Daniel Waltho","doi":"10.1128\/JCM.00662-13","country":"Canada","institute":"McMaster University\/St. Joseph's Healthcare Hamilton","deparment":"Department of Pathology and Molecular Medicine\/Regional Virology and Chlamydiology Laboratory","abstract":"Rapid isothermal amplification methods have recently been introduced, and some of these methods offer significant advantages over PCR. The objective of this study was to develop a rapid and sensitive multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of respiratory syncytial virus subgroups A and B (RSV A and B). We designed six primers each for the matrix gene of RSV A and the polymerase gene of RSV B and developed an M-LAMP assay by using a commercially available master mix and a real-time fluorometer (Genie II; Optigene, United Kingdom) that displays real-time amplification, time to positivity, and amplicon annealing temperature (Tm). The M-LAMP was evaluated against PCR by testing 275 nasopharyngeal (NP) specimens. The final optimized M-LAMP assay had a mean amplification time of 14.2 min (compared with 90 to 120 min for PCR) and had an analytical sensitivity of 1 genome equivalent (ge) for both RSV A and B. Using PCR as a comparator, M-LAMP had a sensitivity of 100% (81\/81) and specificity of 100% (194\/194). We also evaluated a 3- to 10-min specimen processing method involving vortexing with glass beads and heating to 98\u00b0C in M-swab medium (Copan Italia, Brescia, Italy) and found that this rapid processing method allowed detection of 37\/41 (90.2%) of positives when we used extracted nucleic acid. In summary, the M-LAMP assay had excellent sensitivity and specificity for detecting RSV A and B in NP specimens and, when coupled with a rapid specimen preparation method, could provide a specimen-to-result diagnosis time of 30 min.","lamp_id":[{"id":"LPB00066","pathogen":"HRSV A","target":"M"},{"id":"LPB00067","pathogen":"HRSV B","target":"Pol"}]},{"id":40,"pmid":null,"title":"Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP","year":2020,"journal":"medRxiv","authors":"Yinhua Zhang, Nelson Odiwuor, Jin Xiong, Luo Sun, Raphael Ohuru Nyaruaba, Hongping Wei, Nathan A. Tanner","doi":"10.1101\/2020.02.26.20028373","country":"USA","institute":"New England Biolabs","deparment":"","abstract":"The ability to detect an infectious agent in a widespread epidemic is crucial to the success of quarantine efforts in addition to sensitive and accurate screening of potential cases of infection from patients in a clinical setting. Enabling testing outside of sophisticated laboratories broadens the scope of control and surveillance efforts, but also requires robust and simple methods that can be used without expensive instrumentation. Here we report a method to identify SARS-CoV-2 (COVID-19) virus RNA from purified RNA or cell lysis using loop-mediated isothermal amplification (LAMP) using a visual, colorimetric detection. This test was additionally verified using RNA samples purified from respiratory swabs collected from COVID-19 patients in Wuhan, China with equivalent performance to a commercial RT-qPCR test while requiring only heating and visual inspection. This simple and sensitive method provides an opportunity to facilitate virus detection in the field without a requirement for complex diagnostic infrastructure.","lamp_id":[{"id":"LPB00068","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00069","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00070","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00071","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00072","pathogen":"SARS-CoV-2","target":"N"}]},{"id":41,"pmid":35880021,"title":"Competitive SNP-LAMP probes for rapid and robust single-nucleotide polymorphism detection","year":2022,"journal":"Cell Reports Methods","authors":"Leland B. Hyman, Clare R. Christopher, Philip A. Romero","doi":"10.1016\/j.crmeth.2022.100242","country":"","institute":"","deparment":"","abstract":"In this work, we developed a simple and robust assay to rapidly detect SNPs in nucleic acid samples. Our approach combines loop-mediated isothermal amplification (LAMP)-based target amplification with fluorescent probes to detect SNPs with high specificity. A competitive \u201csink\u201d strand preferentially binds to non-SNP amplicons and shifts the free energy landscape to favor specific activation by SNP products. We demonstrated the broad utility and reliability of our SNP-LAMP method by detecting three distinct SNPs across the human genome. We also designed an assay to rapidly detect highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants from crude biological samples. This work demonstrates that competitive SNP-LAMP is a powerful and universal method that could be applied in point-of-care settings to detect any target SNP with high specificity and sensitivity. We additionally developed a publicly available web application for researchers to design SNP-LAMP probes for any target sequence of interest.","lamp_id":[{"id":"LPB00073","pathogen":"SARS-CoV-2","target":"S"}]},{"id":42,"pmid":32900994,"title":"A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics","year":2020,"journal":"Nature Communications","authors":"Michael A Crone, Miles Priestman, Marta Ciechonsk, Kirsten Jensen, David J Sharp, Arthi Anand, Paul Randell, Marko Storch, Paul S Freemont","doi":"10.1038\/s41467-020-18130-3","country":"United Kingdom","institute":"London Biofoundry\/Imperial College London","deparment":"Imperial College Translation and Innovation Hub\/Department of Infectious Disease\/UK Dementia Research Institute Centre for Care Research and Technology","abstract":"The SARS-CoV-2 pandemic has shown how a rapid rise in demand for patient and community sample testing can quickly overwhelm testing capability globally. With most diagnostic infrastructure dependent on specialized instruments, their exclusive reagent supplies quickly become bottlenecks, creating an urgent need for approaches to boost testing capacity. We address this challenge by refocusing the London Biofoundry onto the development of alternative testing pipelines. Here, we present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled. Using an in-house-generated, open-source, MS2-virus-like particle (VLP) SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two detection assays based on CRISPR-Cas13a and RT-loop-mediated isothermal amplification (RT-LAMP). In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and detection of SARS-CoV-2 in patient samples using RT-qPCR, CRISPR-Cas13a, and RT-LAMP. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs, increasing testing capacity by 1000 samples per day.","lamp_id":[{"id":"LPB00074","pathogen":"SARS-CoV-2","target":"N"}]},{"id":43,"pmid":22256968,"title":"Detection of human Enterovirus 71 reverse transcription loop-mediated isothermal amplification (RT-LAMP)","year":2012,"journal":"Letters in Applied Microbiology","authors":"H. Yaqing, Z. Wenping, Y. Zhiyi, W. Xionghu, Y. Shouyi, Y. Hong, D. Yingchun, H. Guifang","doi":"10.1111\/j.1472-765X.2011.03198.x","country":"China","institute":"Southern Medical University","deparment":"Department of Epidemiology","abstract":"Aims: In this study, a one-step, single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and validated for the detection of human Enterovirus 71 (EV71), the major aetiological agent of hand, foot and mouth disease (HFMD).\n\nMethods and results: Reverse transcription loop-mediated isothermal amplification assay was optimized to amplify VP1 gene in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63\u00b0C for 1 h. Amplified products were evaluated by visual inspection and agarose gel electrophoresis. The detection limit of RT-LAMP assay was 10(-5) 100 TCID50 or 160 copies in samples after RNA extraction, which was 10-fold higher in sensitivity than traditional reverse transcription polymerase chain reaction (RT-PCR). The specific positive amplification was only observed in EV71 strains, while no amplification was detected in other tested viruses. Digestion with a specific Escherichia coli restriction enzymes V (EcoR V) demonstrated that the amplified product was unique. A good correlation between RT-LAMP and real-time RT-PCR was observed on the basis of the analysis of 33 clinical samples.\n\nConclusions: Reverse transcription loop-mediated isothermal amplification is a novel, alternative microbiological approach for rapid, sensitive and specific detection of EV71 in HFMD.","lamp_id":[{"id":"LPB00075","pathogen":"EV-A71","target":"VP1"}]},{"id":44,"pmid":33820475,"title":"Rapid visual detection of SARS-CoV-2 by colorimetric loop-mediated isothermal amplification","year":2021,"journal":"Biotechniques","authors":"B\u00e0rbara Reyn\u00e9s, Francisca Serra, Andreu Palou","doi":"10.2144\/btn-2020-0159","country":"Spain","institute":"Universitat de les Illes Balears\/Institut d'Investigaci\u00f3 Sanit\u00e0ria Illes Balears (IdISBa)\/The University of The Balearic Islands","deparment":"Laboratory of Molecular Biology, Nutrition & Biotechnology\/Aliment\u00f3mica S.L.","abstract":"Evaluation of the performance of a new set of primers defined from the ORF1ab sequence, and its combination with a previously published set of primers from the N sequence, to detect SARS-CoV-2 RNA by the loop-mediated isothermal amplification technique is presented. The ORF1ab primer set enables visual detection of SARS-CoV-2 RNA in 16 min. In addition, a simultaneous reaction with both ORF1ab and N primers allows for higher sensitivity of detection, particularly when low numbers of copies are present (250 viral RNA copies). Further, the protocol is able to detect viral RNA in saliva samples. The procedure reported could be easily implemented in the generation of a new and sensitive rapid point-of care device for SARS-CoV-2 RNA visual detection.","lamp_id":[{"id":"LPB00076","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00190","pathogen":"SARS-CoV-2","target":"N"}]},{"id":45,"pmid":27301197,"title":"Rapid and Sensitive Detection of RNA Viruses Based on Reverse Transcription Loop-Mediated Isothermal Amplification, Magnetic Nanoparticles, and Chemiluminescence","year":2016,"journal":"Journal of Biomedical Nanotechnology","authors":"Jiuhai Wang, Peng Lu, Jieni Yan, Yufan Zhang, Lanye Huang, Zeeshan Ali, Zhiyang Li, Nongyue He","doi":"10.1166\/jbn.2016.2244","country":"China","institute":"The Affiliated Drum Tower Hospital of Nanjing University Medical School\/ Southeast University","deparment":"Department of Clinical Laboratory\/State Key Laboratory of Bioelectronics","abstract":"RNA viruses, particularly, the highly pathogenic avian influenza (HPAI) virus, pose serious health concerns, and cause huge economic losses worldwide. Diagnostic tools for the early detection of these deadly RNA viruses are urgently needed to implement treatment and disease control strategies. Conventional reverse transcription polymerase chain reaction (RT-PCR)-based chemiluminescent (RT-PCR-CL) detection is frequently used for the diagnosis of viral infections. However, the requirements for expensive PCR machines and longer thermocycling times are significant drawbacks. In this study, we propose a method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with chemiluminescence (CL) to detect H7N9 virus. The proposed method does not require any expensive instruments, and processing time is remarkably shortened compared to that of RT-PCR-CL. Since several factors including RT-LAMP temperature, probe concentration, hybridization temperature, and hybridization duration might affect the CL signal, each of these parameters was investigated and optimized. One thousand copies\/mL of H7N9 RNA were detectable using the optimized RT-LAMP-CL method. The detection time was significantly reduced by using RT-LAMP, in comparison with conventional RT-PCR-CL. This technique holds great promise for viral detection and diagnosis, especially with regard to avian influenza virus.","lamp_id":[{"id":"LPB00077","pathogen":"Influenza A virus (H7N9)","target":"HA"},{"id":"LPB00078","pathogen":"Influenza A virus (H7N9)","target":"NA"}]},{"id":46,"pmid":25426967,"title":"Integration of reverse transcriptase loop-mediated isothermal amplification with an immunochromatographic strip on a centrifugal microdevice for influenza A virus identification","year":2015,"journal":"Lab on a Chip","authors":"J H Jung, B H Park, S J Oh, G Choi, T S Seo","doi":"10.1039\/c4lc01033g","country":"Republic of Korea","institute":"Korea Advanced Institute of Science and Technology (KAIST)","deparment":"Department of Chemical and Biomolecular Engineering (BK21 Plus program)","abstract":"A novel centrifugal microdevice which could perform reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and immunochromatographic strip (ICS) based amplicon detection was demonstrated for simple and cost-effective influenza A virus identification. The proposed centrifugal microdevice consists of the sample and running buffer loading reservoirs, the RT-LAMP chamber, and the ICS for detecting gene expression. The entire process could be completed sequentially and automatically by simply controlling the rotation speed and by optimizing the microfluidic design. Monoplex and multiplex RT-LAMP reactions targeting H1 and\/or M gene were executed at 66 \u00b0C for 40 min, and the resultant amplicons were successfully analysed on the ICS within 15 min. Influenza A H1N1 virus was subtyped by detecting H1 and M gene on the ICS even with 10 copies of viral RNAs. Highly specific and multiplex viral typing of the integrated RT-LAMP-ICS microdevice was also demonstrated. The combination of the rapid isothermal amplification with the simple colorimetric detection on a strip in a single centrifugal microdevice will provide an advanced genetic analysis platform in the field of on-site pathogen diagnostics.","lamp_id":[{"id":"LPB00079","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00080","pathogen":"Influenza A virus (H3N2)","target":"HA"},{"id":"LPB00081","pathogen":"Influenza A virus (H5N1)","target":"HA"},{"id":"LPB00082","pathogen":"Influenza A virus (H1N1)","target":"M"}]},{"id":47,"pmid":25856093,"title":"Real-time sequence-validated loop-mediated isothermal amplification assays for detection of Middle East respiratory syndrome coronavirus (MERS-CoV)","year":2015,"journal":"PLoS One","authors":"Sanchita Bhadra, Yu Sherry Jiang, Mia R Kumar, Reed F Johnson, Lisa E Hensley, Andrew D Ellington","doi":"10.1371\/journal.pone.0123126","country":"USA","institute":"Institute for Cellular and Molecular Biology","deparment":"Department of Chemistry and Biochemistry","abstract":"The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU\/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.","lamp_id":[{"id":"LPB00083","pathogen":"MERS-CoV","target":"ORF1a"},{"id":"LPB00084","pathogen":"MERS-CoV","target":"ORF1b"},{"id":"LPB00085","pathogen":"MERS-CoV","target":"E"}]},{"id":48,"pmid":28155195,"title":"Clinical evaluation of the isothermal amplification assays for the detection of four common respiratory viruses in children with pneumonia","year":2017,"journal":"Archives of Virology","authors":"Hangyu Zhou, Mengchuan Zhao, Xinna Li, Dan Zhang, Shuaifeng Zhou, Chen Chen, Zhishan Feng, Xuejun Ma","doi":"10.1007\/s00705-017-3227-2","country":"China","institute":"National Institute for Viral Disease Control and Prevention","deparment":"Key Laboratory for Medical Virology","abstract":"Respiratory viruses are recognized as serious causes of morbidity and mortality in lower respiratory tract infections in patients. Isothermal amplification assays are increasingly used in their detection because of their rapidity, simplicity and cost-effectiveness, when compared to traditional molecular diagnostic methods. However, systematic assessment of these assays in the clinical settings is rarely reported. MEDLINE (Pubmed) searches were done analysing subject headings and words in the abstract related to isothermal amplification assay and virus. Selected loop-mediated isothermal amplification assays (LAMP) for respiratory syncytial virus (RSV), human metapneumovirus (HMPV) and adenovirus (ADV) as well as a reverse transcription genome exponential amplification reaction assay (GEAR) for human rhinovirus (HRV) were clinically evaluated in a head to head comparison against a two-tube multiplex reverse transcription-PCR (RT-PCR) assay (two-tube assay) using 634 respiratory specimens from children with pneumonia from different regions in China. Discrepant results between isothermal amplification assays and the two-tube assay were resolved by sequencing. A comparison of sensitivities of each selected isothermal amplification assay among province, gender, and age groups was also analyzed. A total of 634 respiratory specimens selected from Hebei Province children's hospital and Hunan Provincial Center for Disease Control and Prevention were tested. The overall detection rate (number of positive specimens\/total specimens) for viruses tested by Reverse transcription (RT)-LAMP\/LAMP\/RT-GEAR was 35.9% while the detection rate was 46.2% by the two-tube assay. The sensitivity of each isothermal amplification assay was 88.4%, 74.3%, 100% and 73.6% for RSV, HMPV, ADV and HRV, respectively. No false positives were found in isothermal amplification assays. All the discrepant negative results by isothermal amplification assays were confirmed false negatives by sequencing. The LAMP assay for ADV showed significant consistency with the two-tube assay. A higher sensitivity of RSV detection was found in Hunan Province than in Hebei Province (P = 0.01). Among different age groups, a higher sensitivity of RSV detection was also found in children older than 1 year, when compared to children less than 1 year (P = 0.01). The clinical performance of the selected isothermal amplification assays for different viruses varies. Multiple-center assessment is critical to evaluate isothermal amplification assays, especially for RNA viruses, for their broad use in clinical hospital. The selected LAMP assay for the detection of ADV is reliable and has great potential for use in clinics; however, the sensitivities of the LAMP\/GEAR assays for the detection of RSV, HMPV and HRV need to be further improved to meet clinical requirements, although a statistical difference in sensitivity was only found for the selected LAMP assay for RSV.","lamp_id":[{"id":"LPB00086","pathogen":"HRSV A","target":"M"},{"id":"LPB00087","pathogen":"HRSV B","target":"Pol"},{"id":"LPB00088","pathogen":"HAdV","target":"Hexon"},{"id":"LPB00089","pathogen":"HMPV","target":"M"},{"id":"LPB00278","pathogen":"HMPV","target":"M"}]},{"id":49,"pmid":30831121,"title":"Development of real-time fluorescent reverse transcription loop-mediated isothermal amplification assay with quenching primer for influenza virus and respiratory syncytial virus","year":2019,"journal":"Journal of Virological Methods","authors":"Ikuyo Takayama, Mina Nakauchi, Hitoshi Takahashi, Kunihiro Oba, Shohei Semba, Atsushi Kaida, Hideyuki Kubo, Shinji Saito, Shiho Nagat, Takato Odagiri, Tsutomu Kageyama","doi":"10.1016\/j.jviromet.2019.02.010","country":"Japan","institute":"National Institute of Infectious Diseases","deparment":"Influenza Virus Research Center","abstract":"Influenza virus and respiratory syncytial virus cause acute upper and lower respiratory tract infections, especially in children and the elderly. Early treatment for these infections is thought to be important, so simple and sensitive detection methods are needed for use at clinical sites. Therefore, in this study, real-time reverse transcription loop-mediated isothermal amplification assays with quenching primer for influenza virus and respiratory syncytial virus were developed. Evaluation of a total of 113 clinical specimens compared to real-time RT-PCR assays showed that the novel assays could distinguish between the types and subtypes of influenza virus and respiratory syncytial virus and had 100% diagnostic specificity. The diagnostic sensitivity of each assay exceeded 85.0% and the assays showed sufficient clinical accuracy. Furthermore, positive results could be obtained in around 15 min using the novel assays in cases with high concentrations of virus. The developed assays should be useful for identifying influenza virus and respiratory syncytial virus cases not only in experimental laboratories but also in hospital and quarantine laboratories.","lamp_id":[{"id":"LPB00090","pathogen":"Influenza A virus (H3N2)","target":"M"},{"id":"LPB00091","pathogen":"Influenza B virus","target":"NS"},{"id":"LPB00092","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00093","pathogen":"Influenza A virus (H3N2)","target":"HA"},{"id":"LPB00094","pathogen":"RSV A","target":"N"},{"id":"LPB00095","pathogen":"RSV B","target":"N"}]},{"id":50,"pmid":32784757,"title":"SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP","year":2020,"journal":"Viruses","authors":"Steffen Klein, Thorsten G M\u00fcller, Dina Khalid, Vera Sonntag-Buck, Anke-Mareil Heuser, B\u00e4rbel Glass, Matthias Meurer, Ivonne Morales, Angelika Schillak, Andrew Freistaedter, Ina Ambiel , Sophie L Winter, Liv Zimmermann, Tamara Naumoska, Felix Bubeck, Daniel Kirrmaier, Stephanie Ullrich, Isabel Barreto Miranda, Simon Anders, Dirk Grimm, Paul Schnitzler, Michael Knop, Hans-Georg Kr\u00e4usslich, Viet Loan Dao Thi, Kathleen B\u00f6rner, Petr Chlanda","doi":"10.3390\/v12080863","country":"Germany","institute":"Heidelberg University Hospital","deparment":"Center of Infectious Diseases, Virology","abstract":"Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.","lamp_id":[{"id":"LPB00096","pathogen":"SARS-CoV-2","target":"N"}]},{"id":51,"pmid":32915821,"title":"Development of a multiplex isothermal amplification molecular diagnosis method for on-site diagnosis of influenza","year":2020,"journal":"PLoS One","authors":"Woong Sik Jang, Da Hye Lim, Jeonghun Nam, Do-CiC Mihn, Haan Woo Sung, Chae Seung Lim, Jeeyong Kim","doi":"10.1371\/journal.pone.0238615","country":"Republic of Korea","institute":"Korea University Guro Hospital","deparment":"Department of Laboratory Medicine","abstract":"Influenza, which is an acute respiratory disease caused by the influenza virus, represents a worldwide public health and economic problem owing to the significant morbidity and mortality caused by its seasonal epidemics and pandemics. Sensitive and convenient methodologies for the detection of influenza viruses are important for clinical care and infection control as well as epidemiological investigations. Here, we developed a multiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) with quencher\/fluorescence oligonucleotides connected by a 5' backward loop (LF or LB) primer for the detection of two subtypes of influenza viruses: Influenza A (A\/H1 and A\/H3) and influenza B. The detection limits of the multiplex RT-LAMP assay were 10^3 copies and 10^2 copies of RNA for influenza A and influenza B, respectively. The sensitivities of the multiplex influenza A\/B\/IC RT-LAMP assay were 94.62% and 97.50% for influenza A and influenza B clinical samples, respectively. The specificities of the multiplex influenza A\/B\/IC RT-LAMP assay were 100% for influenza A, influenza B, and healthy clinical samples. In addition, the multiplex influenza A\/B\/IC RT-LAMP assay had no cross-reactivity with other respiratory viruses.","lamp_id":[{"id":"LPB00097","pathogen":"Influenza A virus (H3N2)","target":"M"},{"id":"LPB00098","pathogen":"Influenza B virus","target":"NP"}]},{"id":52,"pmid":32883050,"title":"Artificial Intelligence-Assisted Loop Mediated Isothermal Amplification (AI-LAMP) for Rapid Detection of SARS-CoV-2","year":2020,"journal":"Viruses","authors":"Mohammed A Rohaim, Emily Clayton, Irem Sahin, Julianne Vilela, Manar E Khalifa, Mohammad Q Al-Natour, Mahmoud Bayoumi, Aurore C Poirier, Manoharanehru Branavan, Mukunthan Tharmakulasingam, Nouman S Chaudhry, Ravinder Sodi, Amy Brown, Peter Burkhart, Wendy Hacking, Judy Botham, Joe Boyce, Hayley Wilkinson, Craig Williams, Jayde Whittingham-Dowd, Elisabeth Shaw, Matt Hodges, Lisa Butler, Michelle D Bates, Roberto La Ragione, Wamadeva Balachandran, Anil Fernando, Muhammad Munir","doi":"10.3390\/v12090972","country":"United Kingdom","institute":"The Lancaster University","deparment":"Division of Biomedical and Life Sciences","abstract":"Until vaccines and effective therapeutics become available, the practical solution to transit safely out of the current coronavirus disease 19 (CoVID-19) lockdown may include the implementation of an effective testing, tracing and tracking system. However, this requires a reliable and clinically validated diagnostic platform for the sensitive and specific identification of SARS-CoV-2. Here, we report on the development of a de novo, high-resolution and comparative genomics guided reverse-transcribed loop-mediated isothermal amplification (LAMP) assay. To further enhance the assay performance and to remove any subjectivity associated with operator interpretation of results, we engineered a novel hand-held smart diagnostic device. The robust diagnostic device was further furnished with automated image acquisition and processing algorithms and the collated data was processed through artificial intelligence (AI) pipelines to further reduce the assay run time and the subjectivity of the colorimetric LAMP detection. This advanced AI algorithm-implemented LAMP (ai-LAMP) assay, targeting the RNA-dependent RNA polymerase gene, showed high analytical sensitivity and specificity for SARS-CoV-2. A total of ~200 coronavirus disease (CoVID-19)-suspected NHS patient samples were tested using the platform and it was shown to be reliable, highly specific and significantly more sensitive than the current gold standard qRT-PCR. Therefore, this system could provide an efficient and cost-effective platform to detect SARS-CoV-2 in resource-limited laboratories.","lamp_id":[{"id":"LPB00099","pathogen":"SARS-CoV-2","target":"RdRp"}]},{"id":53,"pmid":33635624,"title":"Sequence-Specific Detection of ORF1a, BRAF, and ompW DNA Sequences with Loop Mediated Isothermal Amplification on Lateral Flow Immunoassay Strips Enabled by Molecular Beacons","year":2021,"journal":"Analytical Chemistry","authors":"Marcelino Varona, Derek R Eitzmann, Jared L Anderson","doi":"10.1021\/acs.analchem.0c05355","country":"USA","institute":"Iowa State University","deparment":"Department of Chemistry","abstract":"Loop-mediated isothermal amplification (LAMP) holds great potential for point-of-care (POC) diagnostics due to its speed and sensitivity. However, differentiation between spurious amplification and amplification of the target sequence is a challenge. Herein, we develop the use of molecular beacon (MB) probes for the sequence-specific detection of LAMP on commercially available lateral flow immunoassay (LFIA) strips. The detection of three unique DNA sequences, including ORF1a from SARS-CoV-2, is demonstrated. In addition, the method is capable of detecting clinically relevant single-nucleotide polymorphisms (BRAF V600E). For all sequences tested, the LFIA method offers similar sensitivity to fluorescence detection using a qPCR instrument. We also demonstrate the coupling of the method with solid-phase microextraction to enable isolation and detection of the target sequences from human plasma, pond water, and artificial saliva. Lastly, a 3D printed device is designed and implemented to prevent contamination caused by opening the reaction containers after LAMP.","lamp_id":[{"id":"LPB00100","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":54,"pmid":33806456,"title":"A S imple, A ffordable, R apid, S tabilized, Co lorimetric, V ersatile RT-LAMP Assay to Detect SARS-CoV-2","year":2021,"journal":"Diagnostics","authors":"Juan Garc\u00eda-Bernalt Diego, Pedro Fern\u00e1ndez-Soto, Marta Dom\u00ednguez-Gil, Moncef Belhassen-Garc\u00eda, Juan Luis Mu\u00f1oz Bellido, Antonio Muro","doi":"10.3390\/diagnostics11030438","country":"Spain","institute":"Biomedical Research Institute of Salamanca-Research Centre for Tropical Diseases at the University of Salamanca (IBSAL-CIETUS)","deparment":"Infectious and Tropical Diseases Research Group (e-INTRO)","abstract":"The SARS-CoV-2 pandemic has forced all countries worldwide to rapidly develop and implement widespread testing to control and manage the Coronavirus Disease 2019 (COVID-19). reverse-transcription (RT)-qPCR is the gold standard molecular diagnostic method for COVID-19, mostly in automated testing platforms. These systems are accurate and effective, but also costly, time-consuming, high-technological, infrastructure-dependent, and currently suffer from commercial reagent supply shortages. The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) can be used as an alternative testing method. Here, we present a novel versatile (real-time and colorimetric) RT-LAMP for the simple (one-step), affordable (~1.7 \u20ac\/sample), and rapid detection of SARS-CoV-2 targeting both ORF1ab and N genes of the novel virus genome. We demonstrate the assay on RT-qPCR-positive clinical samples, obtaining most positive results under 25 min. In addition, a novel 30-min one-step drying protocol has been developed to stabilize the RT-LAMP reaction mixtures, allowing them to be stored at room temperature functionally for up to two months, as predicted by the Q10. This Dry-RT-LAMP methodology is suitable for potentially ready-to-use COVID-19 diagnosis. After further testing and validation, it could be easily applied both in developed and in low-income countries yielding rapid and reliable results.","lamp_id":[{"id":"LPB00101","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00175","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00183","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00219","pathogen":"SARS-CoV-2","target":"E"}]},{"id":55,"pmid":32325642,"title":"A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2","year":2020,"journal":"International Journal of Molecular Sciences","authors":"Renfei Lu, Xiuming Wu, Zhenzhou Wan, Yingxue Li, Xia Jin, Chiyu Zhang","doi":"10.3390\/ijms21082826","country":"China","institute":"Nantong Third Hospital Affiliated to Nantong University","deparment":"Clinical Laboratory","abstract":"COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 \u03bcL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 \u03bcL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.","lamp_id":[{"id":"LPB00102","pathogen":"SARS-CoV-2","target":"N"}]},{"id":56,"pmid":32333644,"title":"RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2","year":2020,"journal":"Microbial Biotechnology","authors":"Wei E Huang, Boon Lim, Chia-Chen Hsu, Dan Xiong, Wei Wu, Yejiong Yu, Huidong Jia, Yun Wang, Yida Zeng, Mengmeng Ji, Hong Chang, Xiuming Zhang, Hui Wang, Zhanfeng Cui","doi":"10.1111\/1751-7915.13586","country":"China\/United Kingdom","institute":"University of Oxford","deparment":"Oxford Suzhou Centre for Advanced Research (OSCAR)\/Department of Engineering Science","abstract":"The pandemic coronavirus SARS-CoV-2 in the world has caused a large infected population suffering from COVID-19. To curb the spreading of the virus, WHO urgently demanded an extension of screening and testing; thus, a rapid and simple diagnostic method is needed. We applied a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) to achieve the detection of SARS-CoV-2 in 30 min. We designed four sets of LAMP primers (6 primers in each set), targeting the viral RNA of SARS-CoV-2 in the regions of orf1ab, S gene and N gene. A colorimetric change was used to report the results, which enables the outcome of viral RNA amplification to be read by the naked eye without the need of expensive or dedicated instrument. The sensitivity can be 80 copies of viral RNA per ml in a sample. We validated the RT-LAMP method in a hospital in China, employing 16 clinic samples with 8 positives and 8 negatives. The testing results are consistent with the conventional RT-qPCR. In addition, we also show that one-step process without RNA extraction is feasible to achieve RNA amplification directly from a sample. This rapid, simple and sensitive RT-LAMP method paves a way for a large screening at public domain and hospitals, particularly regional hospitals and medical centres in rural areas.","lamp_id":[{"id":"LPB00103","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00104","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00105","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00106","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":57,"pmid":32300245,"title":"CRISPR\u2013Cas12-based detection of SARS-CoV-2","year":2020,"journal":"Nature Biotechnology","authors":"James P. Broughton, Xianding Deng, Guixia Yu, Clare L. Fasching, Venice Servellita, Jasmeet Singh, Xin Miao, Jessica A. Streithorst, Andrea Granados, Alicia Sotomayor-Gonzalez, Kelsey Zorn, Allan Gopez, Elaine Hsu, Wei Gu, Steve Miller, Chao-Yang Pan, Hugo Guevara, Debra A. Wadford, Janice S. Chen, Charles Y. Chiu","doi":"10.1038\/s41587-020-0513-4","country":"USA","institute":"Mammoth Biosciences\/University of California San Francisco\/UCSF-Abbott Viral Diagnostics and Discovery Center","deparment":"Department of Laboratory Medicine","abstract":"An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with SARS-CoV-2 infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT-PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.","lamp_id":[{"id":"LPB00107","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00302","pathogen":"SARS-CoV-2","target":"E"}]},{"id":58,"pmid":32692666,"title":"Multiplex reverse transcription loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for the diagnosis of COVID-19","year":2020,"journal":"Biosensors and Bioelectronics","authors":"Xiong Zhu, Xiaoxia Wang, Limei Han, Ting Chen, Licheng Wang, Huan Li, Sha Li, Lvfen He, Xiaoying Fu, Shaojin Chen, Mei Xing, Hai Chen, Yi Wang","doi":"10.1016\/j.bios.2020.112437","country":"People's Republic of China","institute":"Central & Clinical Laboratory of Sanya People's Hospital","deparment":"","abstract":"The ongoing global pandemic (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a huge public health issue. Hence, we devised a multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) coupled with a nanoparticle-based lateral flow biosensor (LFB) assay (mRT-LAMP-LFB) for diagnosing COVID-19. Using two LAMP primer sets, the ORF1ab (opening reading frame 1a\/b) and N (nucleoprotein) genes of SARS-CoV-2 were simultaneously amplified in a single-tube reaction, and detected with the diagnosis results easily interpreted by LFB. In presence of FITC (fluorescein)-\/digoxin- and biotin-labeled primers, mRT-LAMP produced numerous FITC-\/digoxin- and biotin-attached duplex amplicons, which were determined by LFB through immunoreactions (FITC\/digoxin on the duplex and anti-FITC\/digoxin on the test line of LFB) and biotin\/treptavidin interaction (biotin on the duplex and strptavidin on the polymerase nanoparticle). The accumulation of nanoparticles leaded a characteristic crimson band, enabling multiplex analysis of ORF1ab and N gene without instrumentation. The limit of detection (LoD) of COVID-19 mRT-LAMP-LFB was 12 copies (for each detection target) per reaction, and no cross-reactivity was generated from non-SARS-CoV-2 templates. The analytical sensitivity of SARS-CoV-2 was 100% (33\/33 oropharynx swab samples collected from COVID-19 patients), and the assay's specificity was also 100% (96\/96 oropharynx swab samples collected from non-COVID-19 patients). The total diagnostic test can be completed within 1 h from sample collection to result interpretation. In sum, the COVID-19 mRT-LAMP-LFB assay is a promising tool for diagnosing SARS-CoV-2 infections in frontline public health field and clinical laboratories, especially from resource-poor regions.","lamp_id":[{"id":"LPB00109","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00110","pathogen":"SARS-CoV-2","target":"N"}]},{"id":59,"pmid":33424386,"title":"Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)-based diagnosis: A potential alternative to quantitative real-time PCR based detection of the novel SARS-COV-2 virus","year":2021,"journal":"Saudi Journal of Biological Sciences","authors":"Farhan Haq, Salmaan Sharif, Adnan Khurshid, Aamer Ikram, Imran Shabbir, Muhammad Salman, Abdul Ahad, Muhammad Suleman Rana, Aroosha Raja, Nazish Badar, Hanaa Tashkandi, Turki Al Amri, Esam I. Azhar, Mohammed S. Almuhayawi, Steve Harakeh, Muhammad Faraz Arshad Malik","doi":"10.1016\/j.sjbs.2020.10.064","country":"Pakistan","institute":"COMSATS University of Islamabad","deparment":"Department of Biosciences","abstract":"The sudden outbreak of the novel Coronavirus infectious disease (COVID-19) resulted in significant challenges to global health systems. One of the primary challenges is rapid, reliable, and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) virus among the suspected COVID-19-infected individuals. At present, quantitative real-time PCR (qRT-PCR) is a widely used diagnostic method. However, it requires expensive instruments and expertise in the interpretation of results. These constraints reflect the significant need for the development of alternative diagnostic options. This study will validate the use and efficiency of the reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay as a potential alternative for the detection of COVID-19. For this purpose, a cohort of 297 suspected COVID-19 patients was tested using both the RT-LAMP assay and the conventional RT-PCR method. For the RT-LAMP assay, three genes (orf-1ab, N, and S) were identified as the target sites for the detection of COVID-19. Based on a comparative assessment, 117 out of 124 positive COVID-19 cases were observed using the RT-LAMP technique with an overall 91.45% sensitivity. Interestingly, where a consensus on 163 individuals free of SARS-Cov-2 was observed, RT-LAMP specificity was 90%. Based on these findings, the robustness of the technique, and the reduced dependency on expensive instrumentation, RT-LAMP-based COVID-19 detection is strongly recommended as a potential alternative assay.","lamp_id":[{"id":"LPB00111","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00297","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00298","pathogen":"SARS-CoV-2","target":"S"}]},{"id":60,"pmid":33712587,"title":"Shotgun transcriptome, spatial omics, and isothermal profiling of SARS-CoV-2 infection reveals unique host responses, viral diversification, and drug interactions","year":2021,"journal":"Nature Communications","authors":"Daniel Butler, Christopher Mozsary, Cem Meydan, Jonathan Foox, Joel Rosiene, Alon Shaiber, David Danko, Ebrahim Afshinnekoo, Matthew MacKay, Fritz J. Sedlazeck, Nikolay A. Ivanov, Maria Sierra, Diana Pohle, Michael Zietz, Undina Gisladottir, Vijendra Ramlall, Evan T. Sholle, Edward J. Schenck, Craig D. Westover, Ciaran Hassan, Krista Ryon, Benjamin Young, Chandrima Bhattacharya, Dianna L. Ng, Andrea C. Granados, Yale A. Santos, Venice Servellita, Scot Federman, Phyllis Ruggiero, Arkarachai Fungtammasan , Chen-Shan Chin, Nathaniel M Pearson, Bradley W Langhorst, Nathan A Tanner, Youngmi Kim, Jason W Reeves, Tyler D Hether, Sarah E Warren, Michael Bailey, Justyna Gawrys, Dmitry Meleshko, Dong Xu, Mara Couto-Rodriguez, Dorottya Nagy-Szakal, Joseph Barrows, Heather Wells, Niamh B O'Hara, Jeffrey A Rosenfeld, Ying Chen, Peter A D Steel, Amos J Shemesh, Jenny Xiang, Jean Thierry-Mieg, Danielle Thierry-Mieg, Angelika Iftner, Daniela Bezdan, Elizabeth Sanchez, Thomas R Campion Jr, John Sipley, Lin Cong, Arryn Craney, Priya Velu, Ari M Melnick, Sagi Shapira, Iman Hajirasouliha, Alain Borczuk, Thomas Iftner, Mirella Salvatore, Massimo Loda, Lars F Westblade, Melissa Cushing, Shixiu Wu, Shawn Levy, Charles Chiu, Robert E Schwartz, Nicholas Tatonetti, Hanna Rennert, Marcin Imielinski, Christopher E Mason","doi":"10.1038\/s41467-021-21361-7","country":"USA","institute":"Weill Cornell Medicine\/New York Genome Center\/Englander Institute for Precision Medicine and the Meyer Cancer Center","deparment":"Department of Physiology and Biophysics\/ The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine\/WorldQuant Initiative for Quantitative Prediction\/ New York Genome Center\/Department of Pathology and Laboratory Medicine","abstract":"In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso\/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin-angiotensin-aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies.","lamp_id":[{"id":"LPB00112","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00113","pathogen":"SARS-CoV-2","target":"N"}]},{"id":61,"pmid":32248677,"title":"Rapid Detection of Influenza Virus Subtypes Based on an Integrated Centrifugal Disc","year":2020,"journal":"ACS Sensors","authors":"Yuhan Yao, Xi Chen, Xinlian Zhang, Qi Liu, Jinhui Zhu, Wang Zhao, Sixiu Liu, Guodong Sui","doi":"10.1021\/acssensors.9b02595","country":"People's Republic of China","institute":"Fudan University","deparment":"Department of Environmental Science and Engineering","abstract":"Influenza is a zoonotic disease, infecting a wide variety of warm-blooded animals. It is caused by an influenza virus, which has been found with hundreds of subtypes. These subtypes are often associated with different sources of infection and possess complex courses of infection. In the early stage of influenza infection, rapid subtype detection is very practicable to prevent the disease from getting worse. Herein, we presented a high-throughput microfluidic centrifugal disc for rapid detection of influenza virus subtypes. The disc realized detection reagent preloads, automated reagent control, and RT-LAMP detections. Six kinds of highly pathogenic influenza viruses could be simultaneously identified, including influenza A subtypes H1, H3, H5, H7, and H9 and influenza B virus. Two different fluorescent dyes could be used on the disc for real-time detection or read by the naked eye. The performance of the disc was demonstrated by testing the clinical samples. The integrated centrifugal disc was expected for rapid detection of influenza virus subtypes to facilitate accurate drug usage in resource-constrained settings and contribute to reduce the risk of the influenza pandemic.","lamp_id":[{"id":"LPB00114","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00115","pathogen":"Influenza A virus (H3N2)","target":"M"},{"id":"LPB00117","pathogen":"Influenza A virus (H5)","target":null},{"id":"LPB00118","pathogen":"Influenza A virus (H7)","target":null},{"id":"LPB00119","pathogen":"Influenza A virus (H9)","target":null},{"id":"LPB00120","pathogen":"Influenza B virus","target":"NS1"}]},{"id":62,"pmid":32680560,"title":"The rapid and visual detection of methicillin-susceptible and methicillin-resistant Staphylococcus aureus using multiplex loop-mediated isothermal amplification linked to a nanoparticle-based lateral flow biosensor","year":2020,"journal":"Antimicrobial Resistance & Infection Control","authors":"Xu Chen, Kai Ma, Xu Yi, Lijuan Xiong, Yu Wang, Shijun Li","doi":"10.1186\/s13756-020-00774-x","country":"","institute":"","deparment":"","abstract":"Background: Staphylococcus aureus (S. aureus), including methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA), is an eminent human pathogen that can colonize the human host and cause severe life-threatening infections. The development of a reliable, simple and rapid assay for detecting S. aureus and identifying MRSA is important for diagnosis and follow-up treatment.\n\nMethods: A novel molecular diagnosis technique, named multiplex loop-mediated isothermal amplification linked to a nanoparticle-based lateral flow biosensor (m-LAMP-LFB), was applied to detect all S. aureus species and identify MRSA. Two sets of primers were designed based on the femA gene (S. aureus-specific gene) and the mecA gene (encoding penicillin-binding protein 2a), and the multiple-LAMP products were analyzed using LFB. The m-LAMP-LFB amplification conditions, including the target DNA concentration, reaction temperature and time, were optimized. The sensitivity and specificity of the m-LAMP-LFB method were tested in the current study, and the multiple-LAMP-LFB technology was applied to detect the MSSA and MRSA strains from clinical samples.\n\nResults: The S. aureus- and MRSA-specific primers based on the femA and mecA genes allowed the multiple-LAMP technology to detect S. aureus and MRSA, respectively. The multiple-LAMP conditions were optimized at 63 \u00b0C for 40 min. The full process, including genomic DNA template preparation, LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the multiple-LAMP assay for femA and mecA detection was 100 fg of genomic DNA template per reaction. The specificity of m-LAMP-LFB detection was 100 %, and no cross-reactions to non-S. aureus strains were observed.\n\nConclusion: The multiple-LAMP-LFB technique developed in the current study is a reliable, simple, rapid, specific and sensitive method to identify MSSA and MRSA infections for appropriate antibiotic therapy.","lamp_id":[{"id":"LPB00116","pathogen":"Staphylococcus aureus","target":"femA"},{"id":"LPB00164","pathogen":"Staphylococcus aureus","target":"mecA"}]},{"id":63,"pmid":32668983,"title":"Direct on-the-spot detection of SARS-CoV-2 in patients","year":2020,"journal":"Experimental Biology and Medicine (Maywood, N.J.)","authors":"Nadav Ben-Assa, Rawi Naddaf, Tal Gefen, Tal Capucha, Haitham Hajjo, Noa Mandelbaum, Lilach Elbaum, Peter Rogov, Daniel A King, Shai Kaplan, Assaf Rotem, Michal Chowers, Moran Szwarcwort-Cohen, Mical Paul, Naama Geva-Zatorsky","doi":"10.1177\/1535370220941819","country":"Israel","institute":"Technion-Institute of Technology","deparment":"Department of Cell Biology and Cancer Science","abstract":"Humanity is currently experiencing a global pandemic with devastating implications on human health and the economy. Most countries are gradually exiting their lockdown state. We are currently lacking rapid and simple viral detections, especially methods that can be performed in the household. Here, we applied RT-LAMP directly on human clinical swabs and self-collected saliva samples. We adjusted the method to allow simple and rapid viral detection, with no RNA purification steps. By testing our method on over 180 human samples, we determined its sensitivity, and by applying it to other viruses, we determined its specificity. We believe this method has a promising potential to be applied world-wide as a simple and cheap surveillance test for SARS-CoV-2.","lamp_id":[{"id":"LPB00121","pathogen":"SARS-CoV-2","target":"N"}]},{"id":64,"pmid":35035293,"title":"Initial evaluation of a mobile SARS-CoV-2 RT-LAMP testing strategy","year":2021,"journal":"Journal of Biomolecular Techniques","authors":"Christina M. Newman, Mitchell D. Ramuta, Matthew T. McLaughlin, Roger W. Wiseman, Julie A. Karl, Dawn M. Dudley, Miranda R. Stauss, Robert J. Maddox, Andrea M. Weiler, Mason I. Bliss, Katrina N. Fauser, Luis A. Haddock, Cecilia G. Shortreed, Amelia K. Haj, Molly A. Accola, Anna S. Heffron, Hailey E. Bussan, Matthew R. Reynolds, Olivia E. Harwood, Ryan V. Moriarty, Laurel M. Stewart, Chelsea M. Crooks, Trent M. Prall, Emma K. Neumann, Elizabeth D. Somsen, Corrie B. Burmeister, Kristi L Hall, William M Rehrauer, Thomas C Friedrich, Shelby L O'Connor, David H O'Connor","doi":"10.7171\/jbt.21-32-03-009","country":"USA","institute":"University of Wisconsin-Madison","deparment":"Pathology and Laboratory Medicine","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control in the United States remains hampered, in part, by testing limitations. We evaluated a simple, outdoor, mobile, colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay workflow where self-collected saliva is tested for SARS-CoV-2 RNA. From July 16, 2020, to November 19, 2020, surveillance samples (n = 4704) were collected from volunteers and tested for SARS-CoV-2 at 5 sites. Twenty-one samples tested positive for SARS-CoV-2 by RT-LAMP; 12 were confirmed positive by subsequent quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, whereas 8 tested negative for SARS-CoV-2 RNA, and 1 could not be confirmed because the donor did not consent to further molecular testing. We estimated the false-negative rate of the RT-LAMP assay only from July 16, 2020, to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or fewer and testing for SARS-CoV-2 RNA by qRT-PCR. We observed a 98.8% concordance between the RT-LAMP and qRT-PCR assays, with only 5 of 421 RT-LAMP-negative pools (2493 total samples) testing positive in the more-sensitive qRT-PCR assay. Overall, we demonstrate a rapid testing method that can be implemented outside the traditional laboratory setting by individuals with basic molecular biology skills and that can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.","lamp_id":[{"id":"LPB00122","pathogen":"SARS-CoV-2","target":"N"}]},{"id":65,"pmid":32937062,"title":"Detection of SARS-CoV-2 with SHERLOCK One-Pot Testing","year":2020,"journal":"The New England Journal of Medicine","authors":"Julia Joung, Alim Ladha, Makoto Saito, Nam-Gyun Kim, Ann E Woolley, Michael Segel, Robert P J Barretto, Amardeep Ranu, Rhiannon K Macrae, Guilhem Faure, Eleonora I Ioannidi, Rohan N Krajeski, Robert Bruneau, Meei-Li W Huang, Xu G Yu, Jonathan Z Li, Bruce D Walker, Deborah T Hung, Alexander L Greninger, Keith R Jerome, Jonathan S Gootenberg, Omar O Abudayyeh, Feng Zhang","doi":"10.1056\/NEJMc2026172","country":"USA","institute":"Massachusetts Institute of Technology","deparment":"","abstract":null,"lamp_id":[{"id":"LPB00123","pathogen":"SARS-CoV-2","target":"N"}]},{"id":66,"pmid":35027870,"title":"Direct detection of SARS-CoV-2 RNA using high-contrast pH-sensitive dyes","year":2021,"journal":"Journal of Biomolecular Techniques","authors":"Timothy A. Brown, Katherine S. Schaefer, Arthur Tsang, Hyun Ah Yi, Jonathan B. Grimm, Andrew L. Lemire, Fadi M. Jradi, Charles Kim, Kevin McGowan, Kimberly Ritola, Derek T. Armstrong, Heba H. Mostafa, Wyatt Korff, Ronald D. Vale, Luke D. Lavis","doi":"10.7171\/jbt.21-3203-007","country":"USA","institute":"Howard Hughes Medical Institute","deparment":"Janelia Research Campus","abstract":"The worldwide coronavirus disease 2019 pandemic has had devastating effects on health, healthcare infrastructure, social structure, and economics. One of the limiting factors in containing the spread of this virus has been the lack of widespread availability of fast, inexpensive, and reliable methods for testing of individuals. Frequent screening for infected and often asymptomatic people is a cornerstone of pandemic management plans. Here, we introduce 2 pH-sensitive \"LAMPshade\" dyes as novel readouts in an isothermal Reverse Transcriptase Loop-mediated isothermal AMPlification amplification assay for severe acute respiratory syndrome coronavirus 2 RNA. The resulting JaneliaLAMP assay is robust, simple, inexpensive, and has low technical requirements, and we describe its use and performance in direct testing of contrived and clinical samples without RNA extraction.","lamp_id":[{"id":"LPB00124","pathogen":"SARS-CoV-2","target":"N"}]},{"id":67,"pmid":21108334,"title":"Evaluation of Reverse Transcription Loop-Mediated Isothermal Amplification Assays for Rapid Diagnosis of Pandemic Influenza A\/H1N1 2009 Virus","year":2010,"journal":"Journal of Medical Virology","authors":"Mina Nakauchi, Tetsushi Yoshikawa, Hidetaka Nakai, Ken Sugata, Akiko Yoshikawa, Yoshizo Asano, Masaru Ihira, Masato Tashiro, Tsutomu Kageyama","doi":"10.1002\/jmv.21934","country":"Japan","institute":"National Institute of Infectious Diseases","deparment":"Influenza Virus Research Center","abstract":"Two genetic diagnosis systems using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technology were evaluated: one for detecting the HA gene of the pandemic influenza A\/H1N1 2009 virus (H1pdm RT-LAMP) and the other for detecting the matrix gene of the influenza A virus (TypeA RT-LAMP). The competence of these two RT-LAMP assay kits for the diagnosis of the pandemic influenza A\/H1N1 2009 virus was compared using real-time RT-PCR assays developed recently on viruses isolated and clinical specimens collected from patients with suspected infection. TypeA RT-LAMP and H1pdm RT-LAMP showed almost the same sensitivity as real-time RT-PCR for viruses isolated. The sensitivity and specificity of TypeA RT-LAMP and H1pdm RT-LAMP were 96.3% and 88.9%, respectively, for clinical specimens. Considering that the ability of the two RT-LAMP assay kits for detection of the pandemic influenza A\/H1N1 2009 virus was comparable to that of the real-time RT-PCR assays, and that the assays were completed within 1 hr and did not require any expensive equipment, these two RT-LAMP assays are promising rapid diagnostic tests for the pandemic influenza A\/H1N1 2009 virus at the hospital bedside.","lamp_id":[{"id":"LPB00125","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00214","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00232","pathogen":"Influenza A virus (H3N2)","target":"M"}]},{"id":68,"pmid":25569879,"title":"Integrated centrifugal reverse transcriptase loop-mediated isothermal amplification microdevice for influenza A virus detection","year":2015,"journal":"Biosensors and Bioelectronics","authors":"Jae Hwan Jung, Byung Hyun Park, Seung Jun Oh, Goro Choi, Tae Seok Seo","doi":"10.1016\/j.bios.2014.12.043","country":"Republic of Korea","institute":"Korea Advanced Institute of Science and Technology (KAIST)","deparment":"Department of Chemical and Biomolecular Engineering (BK21 PLUS Program)","abstract":"An integrated reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) microdevice which consists of microbead-assisted RNA purification and RT-LAMP with real-time monitoring by a miniaturized optical detector was demonstrated. The integrated RT-LAMP microdevice includes four reservoirs for a viral RNA sample (purified influenza A viral RNA or lysates), a washing solution (70% ethanol), an elution solution (RNase-free water), and an RT-LAMP cocktail, and two chambers (a waste chamber and an RT-LAMP reaction chamber). The separate reservoirs for a washing solution, an elution solution, and an RT-LAMP cocktail were designed with capillary valves for stable storage. Three influenza A virus strains (A\/H1N1, A\/H3N2, and A\/H5N1) were used for RNA templates, and RT-LAMP primer sets were designed to detect hemagglutinin (HA) and conserved M gene. Sequential sample flow to the microbeads for RNA purification was achieved by centrifugal force with optimization of capillary valves and a siphon channel. Furthermore, the purified RNA solution was successfully isolated from the waste solution by changing the rotational direction, and combined with the RT-LAMP cocktail in the RT-LAMP reaction chamber for target gene amplification. Total process from the sample injection to the result was completed in 47 min. Influenza A H1N1 virus was confirmed on the integrated RT-LAMP microdevice even with 10 copies of viral RNAs, which revealed 10-fold higher sensitivity than that of a conventional RT-PCR. Subtyping and specificity test of influenza A H1N1 viral lysates were also performed and clinical samples were successfully genotyped to confirm influenza A virus on our proposed integrated microdevice.","lamp_id":[{"id":"LPB00126","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00127","pathogen":"Influenza A virus (H1N1)","target":"M"}]},{"id":69,"pmid":32751106,"title":"A Rapid, Simple, Inexpensive, and Mobile Colorimetric Assay COVID-19-LAMP for Mass On-Site Screening of COVID-19","year":2020,"journal":"International Journal of Molecular Sciences","authors":"Franklin Wang-Ngai Chow, Tony Tat-Yin Chan, Anthony Raymond Tam, Suhui Zhao, Weiming Yao, Joshua Fung, Flora Ka-Kei Cheng, George Chi-Shing Lo, Stella Chu, Kam Leng Aw-Yong, James Yat-Man Tang, Chi-Ching Tsang, Hayes Kam-Hei Luk, Antonio Cheuk-Pui Wong, Kenneth Sze-Ming Li, Longchao Zhu, Zirong He, Emily Wan Ting Tam, Tom Wai-Hin Chung, Sally Cheuk Ying Wong, Tak-Lun Que, Kitty Sau-Chun Fung, David Christopher Lung, Alan Ka-Lun Wu, Ivan Fan-Ngai Hung, Patrick Chiu-Yat Woo, Susanna Kar-Pui Lau","doi":"10.3390\/ijms21155380","country":"Hong Kong","institute":"The University of Hong Kong","deparment":"Department of Microbiology","abstract":"To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies\/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum\/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional transmission.","lamp_id":[{"id":"LPB00128","pathogen":"SARS-CoV-2","target":"ORF3a-E"}]},{"id":70,"pmid":16616961,"title":"Rapid detection and typing of influenza A and B by loop-mediated isothermal amplification: comparison with immunochromatography and virus isolation","year":2006,"journal":"Journal of Virological Methods","authors":"Masahiro Ito, Masahiro Watanabe, Naoko Nakagawa, Toshiaki Ihara, Yoshinobu Okuno","doi":"10.1016\/j.jviromet.2006.03.003","country":"Japan","institute":"Kobe Institute of Health","deparment":"","abstract":"A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was established for the detection of influenza A virus H1 and H3 subtype strains and influenza B virus strains specifically. The total procedure from RNA extraction to virus typing was completed within 3h. In terms of specificity, the representative AH1, AH3 and B strains were detected only by strain-specific primers respectively. No cross-detection was observed. In terms of sensitivity, virus was detected at a minimum concentration of 10 ffu\/ml. Eighty-three nasopharyngeal aspirates obtained from children diagnosed clinically with influenza were tested by the RT-LAMP assay, along with commercially available immunochromatography rapid diagnostic tests and by virus isolation. Virus was isolated from 78 samples (94%) and the subtype was determined by the hemagglutination inhibition test. Although it took at least 3 days, the detection sensitivity was the best of the three methods. With two rapid assays, the detection sensitivity of the RT-LAMP assay (85.5%) was higher than that of immunochromatography tests (75.9%). In addition, the RT-LAMP assay can be used to differentiate emerging influenza virus subtypes by selecting appropriate primer sets.","lamp_id":[{"id":"LPB00129","pathogen":"Influenza A virus (H1)","target":"HA"},{"id":"LPB00130","pathogen":"Influenza A virus (H3N2)","target":"HA"},{"id":"LPB00131","pathogen":"Influenza B virus","target":"NP"}]},{"id":71,"pmid":29763640,"title":"Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus","year":2018,"journal":"Journal of Virological Methods","authors":"Kazuya Shirato, Shohei Semba, Sherif A El-Kafrawy, Ahmed M Hassan, Ahmed M Tolah, Ikuyo Takayama, Tsutomu Kageyama, Tsugunori Notomi, Wataru Kamitani, Shutoku Matsuyama, Esam Ibraheem Azhar","doi":"10.1016\/j.jviromet.2018.05.006","country":"Japan","institute":"National Institute of Infectious Disease","deparment":"Department of Virology III","abstract":"Clinical detection of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in patients is achieved using genetic diagnostic methods, such as real-time RT-PCR assay. Previously, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of MERS-CoV [Virol J. 2014. 11:139]. Generally, amplification of RT-LAMP is monitored by the turbidity induced by precipitation of magnesium pyrophosphate with newly synthesized DNA. However, this mechanism cannot completely exclude the possibility of unexpected reactions. Therefore, in this study, fluorescent RT-LAMP assays using quenching probes (QProbes) were developed specifically to monitor only primer-derived signals. Two primer sets (targeting nucleocapsid and ORF1a sequences) were constructed to confirm MERS cases by RT-LAMP assay only. Our data indicate that both primer sets were capable of detecting MERS-CoV RNA to the same level as existing genetic diagnostic methods, and that both were highly specific with no cross-reactivity observed with other respiratory viruses. These primer sets were highly efficient in amplifying target sequences derived from different MERS-CoV strains, including camel MERS-CoV. In addition, the detection efficacy of QProbe RT-LAMP was comparable to that of real-time RT-PCR assay using clinical specimens from patients in Saudi Arabia. Altogether, these results indicate that QProbe RT-LAMP assays described here can be used as powerful diagnostic tools for rapid detection and surveillance of MERS-CoV infections.","lamp_id":[{"id":"LPB00132","pathogen":"MERS-CoV","target":"N"},{"id":"LPB00133","pathogen":"MERS-CoV","target":"ORF1a"}]},{"id":72,"pmid":32848011,"title":"Development and Clinical Application of a Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for SARS-CoV-2 Infection","year":2020,"journal":"mSphere","authors":"Xuejiao Hu, Qianyun Deng, Junmin Li, Jierong Chen, Zixia Wang, Xiqin Zhang, Zhixin Fang, Haijian Li, Yunhu Zhao, Pan Yu, Wenmin Li, Xiaoming Wang, Shan Li, Lei Zhang, Tieying Hou","doi":"10.1128\/mSphere.00808-20","country":"People's Republic of China","institute":"Guangdong Provincial People's Hospital\/outh China University of Technology","deparment":"Division of Laboratory Medicine\/MOE International Joint Lab for Synthetic Biology and Medicine","abstract":"The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak urgently necessitates sensitive and convenient COVID-19 diagnostics for the containment and timely treatment of patients. We aimed to develop and validate a novel reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2. Patients with suspected COVID-19 and close contacts were recruited from two hospitals between 26 January and 8 April 2020. Respiratory samples were collected and tested using RT-LAMP, and the results were compared with those obtained by reverse transcription-quantitative PCR (RT-qPCR). Samples yielding inconsistent results between these two methods were subjected to next-generation sequencing for confirmation. RT-LAMP was also applied to an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. Samples were collected from a cohort of 129 cases (329 nasopharyngeal swabs) and an independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). The RT-LAMP assay was validated to be accurate (overall sensitivity and specificity of 88.89% and 99.00%, respectively) and diagnostically useful (positive and negative likelihood ratios of 88.89 and 0.11, respectively). RT-LAMP showed increased sensitivity (88.89% versus 81.48%) and high consistency (kappa, 0.92) compared to those of RT-qPCR for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection. The time required for RT-LAMP was less than 1 h from sample preparation to the result. In addition, RT-LAMP was feasible for use with asymptomatic patients and did not cross-react with other respiratory pathogens. The developed RT-LAMP assay offers rapid, sensitive, and straightforward detection of SARS-CoV-2 infection and may aid the expansion of COVID-19 testing in the public domain and hospitals.","lamp_id":[{"id":"LPB00134","pathogen":"SARS-CoV-2","target":"S"}]},{"id":73,"pmid":20381535,"title":"Visual detection of pandemic influenza A H1N1 Virus 2009 by reverse-transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye","year":2010,"journal":"Journal of Virological Methods","authors":"Xue-Jun Ma, Yue-Long Shu, Kai Nie, Meng Qin, Da-Yan Wang, Rong-Bao Gao, Miao Wang, Le-Ying Wen, Feng Han, Shu-Mei Zhou, Xiang Zhao, Yan-Hui Cheng, De-Xin Li, Xiao-Ping Dong","doi":"10.1016\/j.jviromet.2010.03.027","country":"China","institute":"Chinese Center for Disease Control and Prevention","deparment":"National Institute for Viral Disease Control and Prevention","abstract":"A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of pandemic influenza A H1N1 virus infection. The reaction was performed in one step in a single tube at 65 degrees C for 60 min with the addition of hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was approximately 60 copies, and no cross-detection was observed. The assay was evaluated further with 50 clinical specimens diagnosed clinically with seasonal influenza or pandemic influenza A H1N1 virus infection. RT-LAMP with HNB dye was demonstrated to be a sensitive and easy assay for rapid detection of pandemic influenza A H1N1 virus.","lamp_id":[{"id":"LPB00136","pathogen":"Influenza A virus (H1N1)","target":"HA"}]},{"id":74,"pmid":21289470,"title":"Evaluation of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a screening method for the detection of influenza viruses in the fecal materials of water birds","year":2011,"journal":"Journal of Veterinary Medical Science","authors":"Hiromi Yoshida, Yoshihiro Sakoda, Mayumi Endo, Masayuki Motoshima, Fumi Yoshino, Naoki Yamamoto, Masatoshi Okamatsu, Takahiro Soejima, Syouhei Senba, Hidetoshi Kanda, Hiroshi Kida","doi":"10.1292\/jvms.10-0505","country":"Japan","institute":"Hokkaido University","deparment":"Department of Disease Control","abstract":"Migratory water birds are a natural reservoir for influenza A viruses. Viruses replicate in the intestines of ducks and are shed with the fecal materials. Virus isolation from collected fecal materials, therefore, is an integral part of the surveillance of avian influenza in water birds. In the present study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) was assessed for its usefulness in detecting the RNA of influenza A viruses in fecal materials. It was found that, RT-LAMP specifically and sensitively detects the matrix gene of influenza A viruses. Influenza A viruses were isolated from the fecal materials in which viral RNA were detected by RT-LAMP in 35 min. The present findings indicate that RT-LAMP is useful as a high throughput screening method for field samples prior to virus isolation, allowing the processing of hundreds of samples per day.","lamp_id":[{"id":"LPB00137","pathogen":"Influenza A virus (H3N2)","target":"M"}]},{"id":75,"pmid":null,"title":"Rapid detection of SARS-CoV-2 and other respiratory viruses by using LAMP method with Nanopore Flongle workflow","year":2020,"journal":"bioRxiv","authors":"Jingjing Li, Weipeng Quan, Shuge Yan, Shuangju Wu, Jianhu Qin, Tingting Yang, Fan Liang, Depeng Wang, Yu Liang","doi":"10.1101\/2020.06.03.131474","country":"China","institute":"GrandOmics Diagnostics","deparment":"","abstract":"The ongoing novel coronavirus (COVID-19) outbreak as a global public health emergency infected by SARC-CoV-2 has caused devastating loss around the world. Currently, a lot of diagnosis methods have been used to detect the infection. The nucleic acid (NA) testing is reported to be the clinical standard for COVID-19 infection. Evidence shows that a faster and more convenient method to detect in the early phase will control the spreading of SARS-CoV-2. Here, we propose a method to detect SARC-Cov-2 infection within two hours combined with Loop-mediated Isothermal Amplification (LAMP) reaction and nanopore Flongle workflow. In this approach, RNA reverse transcription and nucleic acid amplification reaction with one step in 30 minutes at 60-65\u00b0C constant temperature environment, nanopore Flongle rapidly adapter ligated within 10 minutes. Flongle flow cell sequencing and analysis in real-time. This method described here has the advantages of rapid amplification, convenient operation and real-time detection which is the most important for rapid and reliable clinical diagnosis of COVID-19. Moreover, this approach not only can be used for SARS-CoV-2 detection but also can be extended to other respiratory viruses and pathogens.","lamp_id":[{"id":"LPB00138","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00139","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00140","pathogen":"Influenza B virus","target":"HA"},{"id":"LPB00141","pathogen":"Influenza B virus","target":"M"}]},{"id":76,"pmid":33510181,"title":"Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction","year":2021,"journal":"Scientific Reports","authors":"Shan Wei, Esther Kohl, Alexandre Djandji, Stephanie Morgan, Susan Whittier, Mahesh Mansukhani, Eldad Hod, Mary D\u2019Alton, Yousin Suh, Zev Williams","doi":"10.1038\/s41598-021-81487-y","country":"USA","institute":"Columbia University Medical Center","deparment":"Department of Obstetrics and Gynecology","abstract":"The COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies\/\u03bcl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17\/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.","lamp_id":[{"id":"LPB00142","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":77,"pmid":33479389,"title":"Development and comparison of novel multiple cross displacement amplification (MCDA) assays with other nucleic acid amplification methods for SARS-CoV-2 detection","year":2021,"journal":"Scientific Reports","authors":"Laurence Don Wai Luu, Michael Payne, Xiaomei Zhang, Lijuan Luo, Ruiting Lan","doi":"10.1038\/s41598-021-81518-8","country":"Australia","institute":"University of New South Wales","deparment":"School of Biotechnology and Biomolecular Sciences","abstract":"The development of alternative isothermal amplification assays including multiple cross displacement amplification (MCDA) may address speed and portability limitations of real-time PCR (rt-PCR) methods for SARS-CoV-2 detection. We developed a novel SARS-CoV-2 MCDA assay and compared its speed and sensitivity to loop-mediated isothermal amplification (LAMP) and rt-PCR. Two MCDA assays targeting SARS-CoV-2 N gene and ORF1ab were designed. The fastest time to detection and sensitivity of MCDA was compared to LAMP and rt-PCR using DNA standards and transcribed RNA. For the N gene, MCDA was faster than LAMP and rt-PCR by 10 and 20 min, respectively with fastest time to detection at 5.2 min. rt-PCR had the highest sensitivity with the limit of detection at 10 copies\/\u00b5l compared with MCDA (100 copies\/\u00b5l) and LAMP (500 copies\/\u00b5l). For ORF1ab, MCDA and LAMP had similar speed with fastest time to detection at 9.7 and 8.4 min, respectively. LAMP was more sensitive for ORF1ab detection with 50 copies\/\u00b5l compared to MCDA (500 copies\/\u00b5l). In conclusion, different nucleic acid amplification methods provide different advantages. MCDA is the fastest nucleic acid amplification method for SARS-CoV-2 while rt-PCR is the most sensitive. These advantages should be considered when determining the most suitable nucleic acid amplification methods for different applications.","lamp_id":[{"id":"LPB00143","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00144","pathogen":"SARS-CoV-2","target":"N"}]},{"id":78,"pmid":null,"title":"One-pot Detection of COVID-19 with Real-time Reverse-transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assay and Visual RT-LAMP Assay","year":2020,"journal":"bioRxiv","authors":"Deguo Wang","doi":"10.1101\/2020.04.21.052530","country":"China","institute":"Xuchang University","deparment":"Key Laboratory of Biomarker Based Rapid-detection Technology for Food Safety of Henan Province","abstract":"Background Rapid and reliable diagnostic assays were critical for prevention and control of the coronavirus pneumonia caused by COVID-19.\n\nObjective This study was to establish one-pot real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay and one-pot visual RT-LAMP assay for the detection of COVID-19.\n\nMethods Six specific LAMP primers targeting the N gene of COVID-19 were designed, the RT-LAMP reaction system was optimized with plasmid pUC57 containing N gene sequence, the detection limit was determined with a serial dilution of the plasmid pUC57 containing N gene sequence, and the one-pot real-time RT-LAMP assay and one-pot visual RT-LAMP assay for the detection of COVID-19 were established.\n\nResults Our results showed that the one-pot RT-LAMP assays can detect COVID-19 with a limit of \u2265 6 copies per \u03bcl\u22121 of pUC57 containing N gene sequence.\n\nConclusion This study provides rapid, reliable and sensitive tools for facilitating preliminary and cost-effective prevention and control of COVID-19.","lamp_id":[{"id":"LPB00145","pathogen":"SARS-CoV-2","target":"N"}]},{"id":79,"pmid":33636553,"title":"Extraction-free RT-LAMP to detect SARS-CoV-2 is less sensitive but highly specific compared to standard RT-PCR in 101 samples","year":2021,"journal":"Journal of Clinical Virology","authors":"John J. Schellenberg, Margaret Ormond, Yoav Keynan","doi":"10.1016\/j.jcv.2021.104764","country":"Canada","institute":"Lamp Diagnostics","deparment":"","abstract":"The current scale of public and private testing cannot be expected to meet the emerging need for higher levels of community-level and repeated screening of asymptomatic Canadians for SARS-CoV-2. Rapid point-of-care techniques are increasingly being offered to fill the gap in screening levels required to identify undiagnosed individuals with high viral loads. However, rapid, point-of-care tests often have lower sensitivity in practice. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 has proven sensitive and specific and provides visual results in minutes. Using a commercially available kit for RT-LAMP and primer set targetting nucleocapsid (N), we tested a blinded set of 101 archived nasopharyngeal (NP) swab samples with known RT-PCR results. RT-LAMP reactions were incubated at 65 \u00b0C for 30 min, using heat-inactivated nasopharyngeal swab sample in viral transport medium, diluted tenfold in water, as input. RT-LAMP agreed with all RT-PCR defined negatives (N = 51), and all positives with cycle threshold (Ct) less than 20 (N = 24), 65% of positives with Ct between 20-30 (N = 17), and no positives with Ct greater than 30 (N = 9). RT-LAMP requires fewer and different core components, so may not compete directly with the mainline testing workflow, preserving precious central laboratory resources for those with the greatest need. Careful messaging must be provided when using less-sensitive tests, so that people are not falsely reassured by negative results, but this caveat must be weighed against the clear benefits of reliably identifying those with high levels of virus in prioritized samples at the point of care.","lamp_id":[{"id":"LPB00146","pathogen":"SARS-CoV-2","target":"N"}]},{"id":80,"pmid":null,"title":"Low-Cost Manually Assembled Open Source Reader for Isothermal Pathogen Detection from Saliva using RT-LAMP: SARS-CoV-2 Use Case","year":2020,"journal":"medRxiv","authors":"John C. Bramley, Jason E. Waligorski, Colin L. Kremitzki, Mariel J. Liebeskind, Alex L. Yenkin, Xinyuan E. Xu, Matthew A. Lalli, Jane A. O\u2019Halloran, Philip A. Mudd, Stacey L. House, Robi D. Mitra, Jeffrey D. Milbrandt, William J. Buchser","doi":"10.1101\/2020.10.19.20215319","country":"USA","institute":"Washington University in St Louis School of Medicine","deparment":"","abstract":"Distributed \u201cPoint-of-Care\u201d or \u201cat-Home\u201d testing is an important component for a complete suite of testing solutions. This manuscript describes the construction and operation of a platform technology designed to meet this need. The ongoing COVID-19 pandemic will be used as the proof-of-concept for the efficacy and deployment of this platform. The technology outlined consists of a one-pot, reverse-transcription loop-mediated isothermal amplification (RT-LAMP) chemistry coupled with a low-cost and user-assembled reader using saliva as input. This platform is readily adapted to a wide range of pathogens due to the genetic basis of the reaction. A complete guide to the construction of the reader as well as the production of the reaction chemistry are provided here. Additionally, analytical limit of detection data and the results from saliva testing of SARS-CoV-2, are presented. The platform technology outlined here demonstrates a rapid, distributed, molecular point-of-care solution for pathogen detection using crude sample input.","lamp_id":[{"id":"LPB00147","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00148","pathogen":"SARS-CoV-2","target":"E"}]},{"id":81,"pmid":30696841,"title":"Rapid diagnosis of Mycoplasma pneumonia infection by denaturation bubble-mediated strand exchange amplification: comparison with LAMP and real-time PCR","year":2019,"journal":"Scientific Reports","authors":"Wenqiang Shi, Manman Wei, Qing Wang, Hongwei Wang, Cuiping Ma, Chao Shi","doi":"10.1038\/s41598-018-36751-z","country":"","institute":"","deparment":"","abstract":"M. pneumoniae infection is often ignored due to its similar clinical symptom with respiratory tract infections caused by bacteria or viruses, and thus leading to misdiagnosis and delayed treatment. It is critical to develop a rapid, sensitive and specific diagnosis method. Denaturation Bubble-mediated Strand Exchange Amplification (SEA) was established, which is an isothermal method with only a primer pair and one Bst DNA polymerase. Notably, colorimetric SEA assay was developed with simple visual readout, making instrument-independent in detection step. The method could detect as low as 1.0 \u00d7 10^4 copies\/mL genomic DNA within 60 min. Considering that more than 80% infected patients have 1.0 \u00d7 10^5-1.0 \u00d7 10^7 copies\/mL M. pneumonia DNA, SEA is available for the practical diagnosis of M. pneumoniae in clinical specimens. Through comparing 224 sputum specimens, excellent performance of SEA assay with 90.48% sensitivity and 100% specificity relative to real-time PCR was observed. Compared with LAMP, a comparable sensitivity and low false positive rate was observed for SEA method. Therefore, SEA is a promising method for detecting M. pneumoniae directly from clinical specimens, which is especially suitable for point-of-care testing in primary care facilities and resource-limited settings with minimal equipment and technological expertises.","lamp_id":[{"id":"LPB00150","pathogen":"Mycoplasma pneumoniae","target":"CARDS toxin"}]},{"id":82,"pmid":33165486,"title":"Colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a visual diagnostic platform for the detection of the emerging coronavirus SARS-CoV-2","year":2020,"journal":"Analyst","authors":"Kawin Nawattanapaiboon, Ekawat Pasomsub, Photchanathorn Prombun, Akanit Wongbunmak, Akarawit Jenjitwanich, Pantanat Mahasupachai, Purichaya Vetcho, Cholticha Chayrach, Natthapon Manatjaroenlap, Chonchanok Samphaongern, Treewat Watthanachockchai, Phonthanat Leedorkmai, Suwimon Manopwisedjaroen, Radeekorn Akkarawongsapat, Arunee Thitithanyanont, Matthew Phanchana, Watanalai Panbangred, Somchai Chauvatcharin, Toemsak Srikhirin","doi":"10.1039\/d0an01775b","country":"Thailand","institute":"Zenostic Co.\/Mahidol University","deparment":"School of Materials Science and Innovation","abstract":"COVID-19, caused by the infection of SARS-CoV-2, has emerged as a rapidly spreading infection. The disease has now reached the level of a global pandemic and as a result a more rapid and simple detection method is imperative to curb the spread of the virus. We aimed to develop a visual diagnostic platform for SARS-CoV-2 based on colorimetric RT-LAMP with levels of sensitivity and specificity comparable to that of commercial qRT-PCR assays. In this work, the primers were designed to target a conserved region of the RNA-dependent RNA polymerase gene (RdRp). The assay was characterized for its sensitivity and specificity, and validated with clinical specimens collected in Thailand. The developed colorimetric RT-LAMP assay could amplify the target gene and enabled visual interpretation in 60 min at 65 \u00b0C. No cross-reactivity with six other common human respiratory viruses (influenza A virus subtypes H1 and H3, influenza B virus, respiratory syncytial virus types A and B, and human metapneumovirus) and five other human coronaviruses (MERS-CoV, HKU-1, OC43, 229E and NL63) was observed. The limit of detection was 25 copies per reaction when evaluated with contrived specimens. However, the detection rate at this concentration fell to 95.8% when the incubation time was reduced from 60 to 30 min. The diagnostic performance of the developed RT-LAMP assay was evaluated in 2120 clinical specimens and compared with the commercial qRT-PCR. The results revealed high sensitivity and specificity of 95.74% and 99.95%, respectively. The overall accuracy of the RT-LAMP assay was determined to be 99.86%. In summary, our results indicate that the developed colorimetric RT-LAMP provides a simple, sensitive and reliable approach for the detection of SARS-CoV-2 in clinical samples, implying its beneficial use as a diagnostic platform for COVID-19 screening.","lamp_id":[{"id":"LPB00151","pathogen":"SARS-CoV-2","target":"RdRp"}]},{"id":83,"pmid":33147214,"title":"Development and evaluation of a rapid and simple diagnostic assay for COVID-19 based on loop-mediated isothermal amplification","year":2020,"journal":"PLOS Neglected Tropical Diseases","authors":"Rokusuke Yoshikawa , Haruka Abe, Yui Igasaki, Saeki Negishi, Hiroaki Goto, Jiro Yasuda","doi":"10.1371\/journal.pntd.0008855","country":"Japan","institute":"Nagasaki University\/Institute of Tropical Medicine (NEKKEN)","deparment":"National Research Center for the Control and Prevention of Infectious Diseases (CCPID)\/Department of Emerging Infectious Diseases","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic novel coronavirus that has caused a worldwide outbreak. Here we describe a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that uses a portable device for efficient detection of SARS-CoV-2. This RT-LAMP assay specifically detected SARS-CoV-2 without cross-reacting with the most closely related human coronavirus, SARS-CoV. Clinical evaluation of nasal swab samples from suspected SARS-CoV-2 pneumonia (COVID-19) patients showed that the assay could detect over 23.7 copies within 15 min with a 100% probability. Since the RT-LAMP assay can be performed with a portable battery-supported device, it is a rapid, simple, and sensitive diagnostic assay for COVID-19 that can be available at point-of-care. We also developed the RT-LAMP assay without the RNA extraction step-Direct RT-LAMP, which could detect more than 1.43 x 10^3 copies within 15 min with a 100% probability in clinical evaluation test. Although the Direct RT-LAMP assay was less sensitive than the standard RT-LAMP, the Direct RT-LAMP assay can be available as the rapid first screening of COVID-19 in poorly equipped areas, such as rural areas in developing countries.","lamp_id":[{"id":"LPB00152","pathogen":"SARS-CoV-2","target":"ORF1b"},{"id":"LPB00153","pathogen":"SARS-CoV-2","target":"ORF1b"}]},{"id":84,"pmid":33098286,"title":"A Sensitive Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Direct Visual Detection of SARS-CoV-2","year":2020,"journal":"The American Journal of Tropical Medicine and Hygiene","authors":"Yee Ling Lau, Ilyiana Binti Ismail, Nur Izati Binti Mustapa, Meng Yee Lai, Tuan Suhaila Tuan Soh, Afifah Haji Hassan, Kalaiarasu M Peariasamy, Yee Leng Lee, Pik Pin Goh","doi":"10.4269\/ajtmh.20-1079","country":"Malaysia","institute":"University of Malaya","deparment":"Department of Parasitology","abstract":"A simple and rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of SARS-CoV-2. The RT-LAMP assay was highly specific for SARS-CoV-2 and was able to detect one copy of transcribed SARS-CoV-2 RNA within 24 minutes. Assay validation performed using 50 positive and 32 negative clinical samples showed 100% sensitivity and specificity. The RT-LAMP would be valuable for clinical diagnosis and epidemiological surveillance of SARS-CoV-2 infection in resource-limited areas as it does not require the use of sophisticated and costly equipment.","lamp_id":[{"id":"LPB00154","pathogen":"SARS-CoV-2","target":"N"}]},{"id":85,"pmid":33098427,"title":"Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric reverse-transcription loop-mediated isothermal amplification","year":2020,"journal":"Clinical Chemistry","authors":"Matthew A. Lalli, Joshua S Langmade, Xuhua Chen, Catrina C. Fronick, Christopher S. Sawyer, Lauren C. Burcea, Michael N. Wilkinson, Robert S. Fulton, Michael Heinz, William J. Buchser, Richard D. Head, Robi D. Mitra, Jeffrey Milbrandt","doi":"10.1093\/clinchem\/hvaa267","country":"USA","institute":"Washington University School of Medicine","deparment":"Department of Genetics","abstract":"Background: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor.\n\nMethods: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples.\n\nResults: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%.\n\nConclusions: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.","lamp_id":[{"id":"LPB00155","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00156","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00157","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00158","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"},{"id":"LPB00159","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00353","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00354","pathogen":"SARS-CoV-2","target":"E"}]},{"id":86,"pmid":null,"title":"SLAMP: A Rapid Fluorometric RT-LAMP Assay for Sensitive and Specific Detection of SARS-CoV-2 from Human Saliva","year":2021,"journal":"medRxiv","authors":"D. A. Bikos, C. Hwang, K. A. Brileya, A. Parker, E. K. Loveday, M. Rodriguez, T. LeFevre, I. Thornton, J. N. Wilking, M. Dills, S. T. Walk, A. K. Adams, R. Plowright, A. B. Hoegh, J. R. Carter, J. Morrow, M. Taylor, D. Keil, M. W. Fields, C. B. Chang","doi":"10.1101\/2021.03.31.21254634","country":"USA","institute":"Montana State University","deparment":"Department of Chemical and Biological Engineering\/Center for Biofilm Engineering","abstract":"Rapid testing methods can identify outbreaks and trigger preventive strategies for slowing the spread of SARS-CoV-2, the virus that causes COVID-19. The \u201cgold-standard\u201d detection method for SARS-CoV-2 is reverse transcription quantitative polymerase chain reaction (RT-qPCR) performed on samples collected using a nasopharyngeal (NP) swab. While NP RT-qPCR provides high sensitivity, it requires trained personnel to administer and suffers from lengthy time-to-result. Recently, the testing community has turned to rapid saliva-based screening methods including saliva-to-RT-qPCR and\/or saliva-to-RT-LAMP (reverse transcription loop-mediated isothermal amplification) to identify infected individuals regardless of symptomatic presentation. Here, we report a simple and rapid RT-LAMP fluorometric assay performed directly on heat-inactivated saliva, without the addition of buffers or proteinase K treatments we call saliva LAMP (SLAMP). Over the course of two days, a total of 243 individuals were tested using NP RT-qPCR, saliva-based qPCR, and saliva-based RT-LAMP. Of the 243 NP RT-qPCR tests, 65 were positive, 178 were negative, and SLAMP demonstrated a 91% sensitivity and 98% specificity. SLAMP sensitivity becomes 95% when samples negative in saliva tests while positive in NP RT-qPCR are excluded from evaluation, potentially indicating significant differences in viral titer between collection sites on the body. SLAMP is performed in triplicates and takes 45 min to run in the laboratory, requiring less technician time and instrument run time than NP RT-qPCR. These results demonstrate that saliva-based RT-LAMP can enable frequent and rapid screening of large numbers of people to identify pre-symptomatic and asymptomatic individuals thereby controlling outbreaks.","lamp_id":[{"id":"LPB00160","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00347","pathogen":"SARS-CoV-2","target":"N"}]},{"id":87,"pmid":35128347,"title":"CRISPR-Cas3-based diagnostics for SARS-CoV-2 and influenza virus","year":2022,"journal":"iScience","authors":"Kazuto Yoshimi, Kohei Takeshita, Seiya Yamayoshi, Satomi Shibumura, Yuko Yamauchi, Masaki Yamamoto, Hiroshi Yotsuyanagi, Yoshihiro Kawaoka, Tomoji Mashimo","doi":"10.1016\/j.isci.2022.103830","country":"Japan","institute":"University of Tokyo","deparment":"Division of Animal Genetics\/Division of Genome Engineering","abstract":"CRISPR-based diagnostics (CRISPR-dx), including the Cas12-based DETECTR and Cas13-based SHERLOCK Class 2 CRISPRs, have been used to detect the presence of DNA or RNA from pathogens, such as the 2009 pandemic influenza virus A (IAV) and the 2019 novel coronavirus SARS-CoV-2. Here, we describe the collateral single-stranded DNA cleavage with Class 1 type I CRISPR-Cas3 and highlight its potential for development as a Cas3-mediated rapid (within 40 min), low-cost, instrument-free detection method for SARS-CoV-2. This assay, which we call Cas3-Operated Nucleic Acid detectioN (CONAN), not only detects SARS-CoV-2 in clinical samples, but also offers specific detection of single-base-pair mutations in IAV variants. This tool allows rapid and accurate point-of-care testing for patients with suspected SARS-CoV-2 or drug-resistant IAV infections in hospitals.","lamp_id":[{"id":"LPB00161","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00197","pathogen":"SARS-CoV-2","target":"N"}]},{"id":88,"pmid":33087160,"title":"Rapid point-of-care detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP)","year":2020,"journal":"Virology Journal","authors":"Lena Mautner, Christin-Kirsty Baillie, Heike Marie Herold, Wolfram Volkwein, Patrick Guertler, Ute Eberle, Nikolaus Ackermann, Andreas Sing, Melanie Pavlovic, Ottmar Goerlich, Ulrich Busch, Lars Wassill, Ingrid Huber, Armin Baiker","doi":"10.1186\/s12985-020-01435-6","country":"Germany","institute":"Bavarian Health and Food Safety Authority","deparment":"","abstract":"Background: Fast, reliable and easy to handle methods are required to facilitate urgently needed point-of-care testing (POCT) in the current coronavirus pandemic. Life-threatening severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread all over the world, infecting more than 33,500,000 people and killing over 1 million of them as of October 2020. Infected individuals without any symptoms might still transfer the virus to others underlining the extraordinary transmissibility of this new coronavirus. In order to identify early infections effectively, treat patients on time and control disease spreading, rapid, accurate and onsite testing methods are urgently required.\n\nResults: Here we report the development of a loop-mediated isothermal amplification (LAMP) based method to detect SARS-CoV-2 genes ORF8 and N directly from pharyngeal swab samples. The established reverse transcription LAMP (RT-LAMP) assay detects SARS-CoV-2 directly from pharyngeal swab samples without previous time-consuming and laborious RNA extraction. The assay is sensitive and highly specific for SARS-CoV-2 detection, showing no cross reactivity when tested on 20 other respiratory pathogens. The assay is 12 times faster and 10 times cheaper than routine reverse transcription real-time polymerase chain reaction, depending on the assay used.\n\nConclusion: The fast and easy to handle RT-LAMP assay amplifying specifically the genomic regions ORF8 and N of SARS-CoV-2 is ideally suited for POCT at e.g. railway stations, airports or hospitals. Given the current pandemic situation, rapid, cost efficient and onsite methods like the here presented RT-LAMP assay are urgently needed to contain the viral spread.","lamp_id":[{"id":"LPB00162","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00163","pathogen":"SARS-CoV-2","target":"ORF8"}]},{"id":89,"pmid":33073982,"title":"Rapid Differential Diagnosis of Seven Human Respiratory Coronaviruses Based on Centrifugal Microfluidic Nucleic Acid Assay","year":2020,"journal":"Analytical Chemistry","authors":"Huiwen Xiong, Xin Ye,Yang Li, Lijuan Wang, Jin Zhang, Xueen Fang, Jilie Kong","doi":"10.1021\/acs.analchem.0c03364","country":"People's Republic of China","institute":"Fudan University","deparment":"Department of Chemistry and Institutes of Biomedical Sciences","abstract":"With the global outbreak of the coronavirus disease 2019 (COVID-19), the highly infective, highly pathogenic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has attracted great attention. Currently, a method to simultaneously diagnose the seven known types human coronaviruses remains lacking and is urgently needed. In this work, we successfully developed a portable microfluidic system for the rapid, accurate, and simultaneous detection of SARS-CoV, middle east respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2, and four other human coronaviruses (HCoVs) including HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. The disk-like microfluidic platform integrated with loop-mediated isothermal amplification provides highly accurate, sensitive, and specific results with a wide linear range within 40 min. The diagnostic tool achieved 100% consistency with the \"gold standard\" polymerase chain reaction in detecting 54 real clinical samples. The integrated system, with its simplicity, is urgently needed for the diagnosis of SARS-CoV-2 during the COVID-19 pandemic.","lamp_id":[{"id":"LPB00165","pathogen":"HCoV-229E","target":"N"},{"id":"LPB00166","pathogen":"HCoV-OC43","target":"N"},{"id":"LPB00167","pathogen":"HCoV-NL63","target":"N"},{"id":"LPB00168","pathogen":"HCoV-HKU1","target":"N"},{"id":"LPB00169","pathogen":"SARS-CoV","target":"N"},{"id":"LPB00170","pathogen":"MERS-CoV","target":"N"},{"id":"LPB00171","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00172","pathogen":"SARS-CoV-2","target":"N"}]},{"id":90,"pmid":33148808,"title":"Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2","year":2020,"journal":"Proceedings of the National Academy of Sciences of the United States of America","authors":"Ashwin Ramachandran, Diego A Huyke, Eesha Sharma, Malaya K Sahoo, ChunHong Huang, Niaz Banaei, Benjamin A Pinsky, Juan G Santiago","doi":"10.1073\/pnas.2010254117","country":"USA","institute":"Stanford University","deparment":"Department of Aeronautics & Astronautics","abstract":"The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter nonspecifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by cofocusing Cas12-gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification and the ITP-enhanced CRISPR assay to achieve detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (from raw sample to result) in about 35 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables an alternate modality for a suite of microfluidic CRISPR-based diagnostic assays.","lamp_id":[{"id":"LPB00173","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00174","pathogen":"SARS-CoV-2","target":"E"}]},{"id":91,"pmid":31114822,"title":"Demonstration of a quantitative triplex LAMP assay with an improved probe-based readout for the detection of MRSA","year":2019,"journal":"Analyst","authors":"Imaly A Nanayakkara, Ian M White","doi":"10.1039\/c9an00671k","country":"","institute":"","deparment":"","abstract":"As molecular diagnostics move away from polymerase chain reaction (PCR) in order to target point-of-care testing applications, loop-mediated isothermal amplification (LAMP) is gaining popularity due its rapid, sensitive and specific detection with simpler instrumentation. However, while Taqman PCR enables real-time quantitative readout and multiplexed gene detection in single samples, analogous methods in LAMP are not yet broadly developed. To date, the real-time detection methods applied to LAMP involve turbidimetry or measuring fluorescence of an intercalator; however, both of these methods are nonspecific to the target of interest and do not allow for multiple gene detection in a single sample. Probe-based methods have been developed to address the need for specific target detection and multiplexed, one-pot reactions, but most of these methods have strict assay conditions and require the design of loop primers, which is not always possible. DARQ LAMP is a probe-based method that offers the most promise for quantitative and real-time multiplexed detection, as it has a relatively simple design and can be used in either a four-primer or six-primer system. However, previous work has only shown the assay to function well in a narrow range of reaction conditions, which is restrictive given that various LAMP assays require a broad range of conditions. In this work we investigate the use of the newest-generation strand-displacing polymerase and demonstrate that it has higher tolerance to reaction conditions than previous polymerases. Using the results from these studies, we demonstrate a single-reaction triplex assay for the detection of methicillin-resistant S. aureus (MRSA), which would not be possible with any of the previously reported LAMP systems.","lamp_id":[{"id":"LPB00176","pathogen":"Staphylococcus aureus","target":"femB"},{"id":"LPB00177","pathogen":"Staphylococcus aureus","target":"femB"},{"id":"LPB00178","pathogen":"Staphylococcus aureus","target":"mecA"}]},{"id":92,"pmid":35049639,"title":"Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities","year":2021,"journal":"Biosensors","authors":"Zhijian Yi, Jean de Dieu Habimana, Omar Mukama, Zhiyuan Li, Nelson Odiwuor, Hanzhi Jing, Chengrong Nie, Mei Hu, Zuoxian Lin, Hongping Wei, Lingwen Zeng","doi":"10.3390\/bios12010011","country":"","institute":"","deparment":"","abstract":"Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to a global pandemic with a high spread rate and pathogenicity. Thus, with limited testing solutions, it is imperative to develop early-stage diagnostics for rapid and accurate detection of SARS-CoV-2 to contain the rapid transmission of the ongoing COVID-19 pandemic. In this regard, there remains little knowledge about the integration of the CRISPR collateral cleavage mechanism in the lateral flow assay and fluorophotometer. In the current study, we demonstrate a CRISPR\/Cas12a-based collateral cleavage method for COVID-19 diagnosis using the Cas12a\/crRNA complex for target recognition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) for sensitivity enhancement, and a novel DNA capture probe-based lateral flow strip (LFS) or real-time fluorescence detector as the parallel system readout facility, termed CRICOLAP. Our novel approach uses a customized reporter that hybridizes an optimized complementary capture probe fixed at the test line for naked-eye result readout. The CRICOLAP system achieved ultra-sensitivity of 1 copy\/\u00b5L in ~32 min by portable real-time fluorescence detection and ~60 min by LFS. Furthermore, CRICOLAP validation using 60 clinical nasopharyngeal samples previously verified with a commercial RT-PCR kit showed 97.5% and 100% sensitivity for S and N genes, respectively, and 100% specificity for both genes of SARS-CoV-2. CRICOLAP advances the CRISPR\/Cas12a collateral cleavage result readout in the lateral flow assay and fluorophotometer, and it can be an alternative method for the decentralized field-deployable diagnosis of COVID-19 in remote and limited-resource locations.","lamp_id":[{"id":"LPB00179","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00180","pathogen":"SARS-CoV-2","target":"N"}]},{"id":93,"pmid":34979349,"title":"A Palm Germ-Radar (PaGeR) for rapid and simple COVID-19 detection by reverse transcription loop-mediated isothermal amplification (RT-LAMP)","year":2022,"journal":"Biosensors and Bioelectronics","authors":"Anle Ge, Fengyi Liu, Xindong Teng, Chaojie Cui, Fei Wu, Wenjing Liu, Yang Liu, Xiaoguang Chen, Jian Xu, Bo Ma","doi":"10.1016\/j.bios.2021.113925","country":"","institute":"","deparment":"","abstract":"The current COVID-19 pandemic caused by SARS-CoV-2 is raging, seriously threatening people's lives. The establishment of rapid and accurate pathogen detection technology is not only critical in this epidemic, but also a reminder that we must always be prepared for possible future outbreaks. Therefore, we developed a Palm Germ-Radar (PaGeR) device for rapid and simple detection of COVID-19 from extracted patient sample RNA by RT-LAMP. The whole procedure of rapid COVID-19 detection is based on 4 simple steps: inactivation, extraction, amplification, and detection. SARS-CoV-2 down to 1 copy\/\u03bcL could be detected selectively with naked-eye. Three detection methods (colorimetric, fluorometric and lateral dipstick readout) could be performed in PaGeR instrument. By employing the PaGeR, we successfully detected SARS-CoV-2 in clinical RNA samples isolated from swab specimens. The results showed that 15 out of 17 COVID-19 patients were diagnosed as positive while all 55 normal samples were diagnosed as negative. Therefore, the developed PaGeR instrument can realize the detection of COVID-19 with easily visualized results, providing a promising instrument for rapid detection in the community as well as at home.","lamp_id":[{"id":"LPB00181","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00227","pathogen":"SARS-CoV-2","target":"N"}]},{"id":94,"pmid":33771097,"title":"Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis","year":2021,"journal":"Molecular Medicine","authors":"Bruna de Oliveira Coelho, Heloisa Bruna Soligo Sanchuki, Dalila Luciola Zanette, Jeanine Marie Nardin, Hugo Manuel Paz Morales, Bruna Fornazari, Mateus N\u00f3brega Aoki, Lucas Blanes","doi":"10.1186\/s10020-021-00289-0","country":"Brazil","institute":"Carlos Chagas Institute","deparment":"Laboratory for Applied Science and Technology in Health","abstract":"Background: SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results.\n\nMethods: To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm.\n\nResults: First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high.\n\nConclusion: This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.","lamp_id":[{"id":"LPB00182","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":95,"pmid":33382861,"title":"Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab samples","year":2020,"journal":"PLoS One","authors":"Dawn M. Dudley, Christina M. Newman, Andrea M. Weiler, Mitchell D. Ramuta, Cecilia G. Shortreed, Anna S. Heffron, Molly A. Accola, William M. Rehrauer, Thomas C. Friedrich, David H. O\u2019Connor","doi":"10.1371\/journal.pone.0244882","country":"USA","institute":"University of Wisconsin-Madison","deparment":"Department of Pathology and Laboratory Medicine","abstract":"SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Frequent surveillance of individuals attending work or school is currently unavailable to most people but will likely be necessary to reduce the ~50% of transmission that occurs when individuals are nonsymptomatic. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed primary samples with a limit of detection (LOD) of ~625 copies\/\u03bcl, approximately 100-fold lower sensitivity than qRT-PCR. While less sensitive than extraction-based molecular methods, RT-LAMP without RNA extraction is fast and inexpensive. Here we also demonstrate that adding a lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 surveillance programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.","lamp_id":[{"id":"LPB00184","pathogen":"SARS-CoV-2","target":"N"}]},{"id":96,"pmid":33251206,"title":"Combinations of PCR and Isothermal Amplification Techniques Are Suitable for Fast and Sensitive Detection of SARS-CoV-2 Viral RNA","year":2020,"journal":"Frontiers in Bioengineering and Biotechnology","authors":"Dmitriy A. Varlamov, Konstantin A. Blagodatskikh, Evgenia V. Smirnova, Vladimir M. Kramarov, Konstantin B. Ignatov","doi":"10.3389\/fbioe.2020.604793","country":"Russia","institute":"Syntol JSC","deparment":"","abstract":"The newly identified coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causes coronavirus disease 2019 (COVID-19) and has affected over 25 million people worldwide as of August 31, 2020. To aid in the development of diagnostic kits for rapid and sensitive detection of the virus, we evaluated a combination of polymerase chain reaction (PCR) and isothermal nucleic acid amplification techniques. Here, we compared conventional PCR and loop-mediated isothermal amplification (LAMP) methods with hybrid techniques such as polymerase chain displacement reaction (PCDR) and a newly developed PCR-LAMP method. We found that the hybrid methods demonstrated higher sensitivity and assay reaction rates than those of the classic LAMP and PCR techniques and can be used to for SARS-CoV-2 detection. The proposed methods based on the modern hybrid amplification techniques markedly improve virus detection and, therefore, can be extremely useful in the development of new diagnostic kits.","lamp_id":[{"id":"LPB00185","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":97,"pmid":null,"title":"A simple direct RT-LAMP SARS-CoV-2 saliva diagnostic","year":2020,"journal":"medRxiv","authors":"Michael J. Flynn, Olga Snitser, James Flynn, Samantha Green, Idan Yelin, Moran Szwarcwort-Cohen, Roy Kishony, Michael B. Elowitz","doi":"10.1101\/2020.11.19.20234948","country":"USA","institute":"California Institute of Technology","deparment":"Applied Physics and Materials Science","abstract":"Widespread, frequent testing is essential for curbing the ongoing COVID-19 pandemic. Because its simplicity makes it ideal for widely distributed, high throughput testing, RT-LAMP provides an attractive alternative to RT-qPCR. However, most RT-LAMP protocols require the purification of RNA, a complex and low-throughput bottleneck that has often been subject to reagent supply shortages. Here, we report an optimized RT-LAMP-based SARS-CoV-2 diagnostic protocol for saliva and swab samples. In the protocol we replace RNA purification with a simple sample preparation step using a widely available chelating agent, as well as optimize key protocol parameters. When tested on clinical swab and saliva samples, this assay achieves a limit of detection of 10^5 viral genomes per ml, with sensitivity close to 90% and specificity close to 100%, and takes 45 minutes from sample collection to result, making it well suited for a COVID-19 surveillance program.","lamp_id":[{"id":"LPB00186","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00187","pathogen":"SARS-CoV-2","target":"N"}]},{"id":98,"pmid":34082799,"title":"Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons","year":2021,"journal":"Genome Biology","authors":"Scott Sherrill-Mix, Young Hwang, Aoife M. Roche, Abigail Glascock, Susan R. Weiss, Yize Li, Leila Haddad, Peter Deraska, Caitlin Monahan, Andrew Kromer, Jevon Graham-Wooten, Louis J. Taylor, Benjamin S. Abella, Arupa Ganguly, Ronald G. Collman, Gregory D. Van Duyne, Frederic D. Bushman","doi":"10.1186\/s13059-021-02387-y","country":"USA","institute":"University of Pennsylvania","deparment":"Department of Microbiology\/Department of Medicine","abstract":"Background: Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection.\n\nResults: Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in \"single pot\" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes.\n\nConclusions: LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.","lamp_id":[{"id":"LPB00188","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00189","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00349","pathogen":"SARS-CoV-2","target":"E"}]},{"id":99,"pmid":34930933,"title":"Evaluation of reverse transcription-loop-mediated isothermal amplification for rapid detection of SARS-CoV-2","year":2021,"journal":"Scientific Reports","authors":"Willi Quino, Diana Flores-Le\u00f3n, Junior Caro-Castro, Carmen V. Hurtado, Iris Silva, Ronnie G. Gavilan","doi":"10.1038\/s41598-021-03623-y","country":"","institute":"","deparment":"","abstract":"The main strategy for response and control of COVID-19 demands the use of rapid, accurate diagnostic tests aimed at the first point of health care. During the emergency, an increase in asymptomatic and symptomatic cases results in a great demand for molecular tests, which is promoting the development and application of rapid diagnostic technologies. In this study, we describe the development and evaluation of RT-LAMP to detect SARS-CoV-2 based on three genes (ORF1ab, M and N genes) in monoplex and triplex format. RT-LAMP assays were compared with the gold standard method RT-qPCR. The triplex format (RdRp, M and N genes) allowed obtaining comparable results with de RT-qPCR (RdRp and E genes), presented a sensitivity of 98.9% and a specificity of 97.9%, opening the opportunity to apply this method to detect SARS-CoV-2 at primary health-care centers.","lamp_id":[{"id":"LPB00191","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"},{"id":"LPB00224","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00308","pathogen":"SARS-CoV-2","target":"M"},{"id":"LPB00817","pathogen":"SARS-CoV-2","target":"RdRp"}]},{"id":100,"pmid":33610926,"title":"Development of a point-of-care test to detect SARS-CoV-2 from saliva which combines a simple RNA extraction method with colorimetric reverse transcription loop-mediated isothermal amplification detection","year":2021,"journal":"Journal of Clinical Virology","authors":"Wataru Yamazaki,Yasufumi Matsumura, Uraiwan Thongchankaew-Seo, Yasuko Yamazaki, Miki Nagao","doi":"10.1016\/j.jcv.2021.104760","country":"Japan","institute":"Kyoto University\/Kyoto University Graduate School of Medicine\/\/Kyoto University School of Public Health","deparment":"Center for Southeast Asian Studies","abstract":"The new coronavirus infection (COVID-19) is a major public health concern, with a high burden and risk for infection among patients and healthcare workers. Saliva droplets containing SARS-COV-2 are a major vector for COVID-19 infection, making saliva a promising alternative for COVID-19 testing using nasopharyngeal swab samples. To diagnose COVID-19 patients in the field, a point-of-care test (POCT) using saliva was conceptualized. We have developed a simple method for extracting RNA from saliva samples using semi-alkaline proteinase, a sputum homogenizer typically used for preparing samples for tuberculosis testing, and a subsequent simple heating step with no need for centrifugation or RNA extraction. Further, we newly developed a triplex reverse transcription loop-mediated isothermal amplification approach (RT-LAMP) which utilizes colorimetric readout using a heat block, with results evaluated with the unaided eye. In 44 clinical patients suspected of having COVID-19 infection, the test took 45 min, and resulted in a diagnostic sensitivity of 82.6% (19\/23) and diagnostic specificity of 100% (21\/21), compared to the reference standard. The limit of detection was 250 copies\/reaction (25,000 copies\/mL). Our newly developed POCT approach achieved simple RNA extraction and constant RT-LAMP detection. This POCT has the potential to be used for simple inspection stations in a field setting, helping reduce the risk of infection by simplifying and accelerating testing for COVID-19.","lamp_id":[{"id":"LPB00192","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00193","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00194","pathogen":"SARS-CoV-2","target":"ORF7a"}]},{"id":101,"pmid":33626039,"title":"Ultra-rapid detection of SARS-CoV-2 in public workspace environments","year":2021,"journal":"PLoS One","authors":"Ozlem Yaren, Jacquelyn McCarter, Nikhil Phadke, Kevin M. Bradley, Benjamin Overton, Zunyi Yang, Shatakshi Ranade, Kunal Patil, Rishikesh Bangale, Steven A. Benner","doi":"10.1371\/journal.pone.0240524","country":"USA","institute":"Foundation for Applied Molecular Evolution","deparment":"","abstract":"Managing the pandemic caused by SARS-CoV-2 requires new capabilities in testing, including the possibility of identifying, in minutes, infected individuals as they enter spaces where they must congregate in a functioning society, including workspaces, schools, points of entry, and commercial business establishments. Here, the only useful tests (a) require no sample transport, (b) require minimal sample manipulation, (c) can be performed by unlicensed individuals, (d) return results on the spot in much less than one hour, and (e) cost no more than a few dollars. The sensitivity need not be as high as normally required by the FDA for screening asymptomatic carriers (as few as 10 virions per sample), as these viral loads are almost certainly not high enough for an individual to present a risk for forward infection. This allows tests specifically useful for this pandemic to trade-off unneeded sensitivity for necessary speed, simplicity, and frugality. In some studies, it was shown that viral load that creates forward-infection risk may exceed 10^5 virions per milliliter, easily within the sensitivity of an RNA amplification architecture, but unattainable by antibody-based architectures that simply target viral antigens. Here, we describe such a test based on a displaceable probe loop amplification architecture.","lamp_id":[{"id":"LPB00195","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00409","pathogen":"SARS-CoV-2","target":"N"}]},{"id":102,"pmid":33126177,"title":"opvCRISPR: One-pot visual RT-LAMP-CRISPR platform for SARS-cov-2 detection","year":2021,"journal":"Biosensors and Bioelectronics","authors":"Rui Wang, Chunyan Qian, Yanan Pang, Miaomiao Li, Yu Yang, Haijing Ma, Manying Zhao, Feng Qian, Hang Yu, Zhenping Liu, Ting Ni, Yan Zheng, Yongming Wang","doi":"10.1016\/j.bios.2020.112766","country":"People's Republic of China","institute":"Fudan University","deparment":"Department of Chemistry & State Key Laboratory of Molecular Engineering of Polymers","abstract":"The 2019 novel coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected all aspects of human life. Rapid, accurate, sensitive and user friendly detection method is urgently needed to facilitate early intervention and control the spread of SARS-CoV-2. Here, we propose a one-pot visual SARS-CoV-2 detection system named \"opvCRISPR\" by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Cas12a cleavage in a single reaction system. We demonstrate that the collateral activity against single-stranded DNA (ssDNA) reporters of activated Cas12a triggered by RT-LAMP amplicon increases detection sensitivity and makes detection results observable with naked eye. The opvCRISPR enables detection at nearly single molecule level in 45 min. We validate this method with 50 SARS-CoV-2 potentially infected clinical samples. The opvCRISPR diagnostic results provide 100% agreement with the Centers for Disease Control and Prevention (CDC)-approved quantitative RT-PCR assay. The opvCRISPR holds great potential for SARS-CoV-2 detection in next-generation point-of-care molecular diagnostics.","lamp_id":[{"id":"LPB00196","pathogen":"SARS-CoV-2","target":"S"}]},{"id":103,"pmid":null,"title":"A comparative study of isothermal nucleic acid amplification methods for SARS-CoV-2 detection at point-of-care","year":2020,"journal":"bioRxiv","authors":"Diem Hong Tran, Hoang Quoc Cuong, Hau Thi Tran, Uyen Phuong Le, Hoang Dang Khoa Do, Le Minh Bui, Nguyen Duc Hai, Hoang Thuy Linh, Nguyen Thi Thanh Thao, Nguyen Hoang Anh, Nguyen Trung Hieu, Cao Minh Thang, Van Van Vu, Huong Thi Thu Phung","doi":"10.1101\/2020.05.24.113423","country":"Vietnam","institute":"Nguyen Tat Thanh University","deparment":"NTT Hi-Tech Institute","abstract":"COVID-19, caused by the novel coronavirus SARS-CoV-2, has spread worldwide and put most of the world under lockdown. Despite that there have been emergently approved vaccines for SARS-CoV-2, COVID-19 cases, hospitalizations, and deaths have remained rising. Thus, rapid diagnosis and necessary public health measures are still key parts to contain the pandemic. In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS-CoV-2 detection based on loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and polymerase spiral reaction (PSR) were designed and evaluated. The three methods showed the same limit of detection (LOD) value of 1 copy of the targeted gene per reaction. However, for the direct detection of SARS-CoV-2 genomic-RNA, LAMP outperformed both CPA and PSR, exhibiting the LOD value of roughly 43.14 genome copies\/reaction. The results can be read with the naked eye within 45 minutes, without cross-reactivity to closely related coronaviruses. Moreover, the direct detection of SARS-CoV-2 RNA in simulated patient specimens by iNAATs was also successful. Finally, the ready-to-use lyophilized reagents for LAMP reactions were shown to maintain the sensitivity and LOD value of the liquid assays. The results indicate that the colorimetric lyophilized LAMP kit developed herein is highly suitable for detecting SARS-CoV-2 nucleic acids at point-of-care.","lamp_id":[{"id":"LPB00199","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00200","pathogen":"SARS-CoV-2","target":"N"}]},{"id":104,"pmid":33469065,"title":"Direct detection of SARS-CoV-2 using non-commercial RT-LAMP reagents on heat-inactivated samples","year":2021,"journal":"Scientific Reports","authors":"Alisa Alekseenko, Donal Barrett, Yerma Pareja-Sanchez, Rebecca J. Howard, Emilia Strandback, Henry Ampah-Korsah, Ur\u0161ka Rov\u0161nik, Silvia Zuniga-Veliz, Alexander Klenov, Jayshna Malloo, Shenglong Ye, Xiyang Liu, Bj\u00f6rn Reinius, Simon J. Els\u00e4sser, Tomas Nyman, Gustaf Sandh, Xiushan Yin, Vicent Pelechano","doi":"10.1038\/s41598-020-80352-8","country":"Sweden","institute":"Karolinska Institutet","deparment":"Department of Microbiology, Tumor and Cell Biology","abstract":"RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.","lamp_id":[{"id":"LPB00201","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00202","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":105,"pmid":33497538,"title":"Loop-mediated isothermal amplification for the detection of SARS-CoV-2 in saliva","year":2021,"journal":"Microbial Biotechnology","authors":"Monika Jan\u00edkov\u00e1, J\u00falius Hodosy, Peter Boor, Boris Klempa, Peter Celec","doi":"10.1111\/1751-7915.13737","country":"Slovakia","institute":"Comenius University","deparment":"Institute of Molecular Biomedicine","abstract":"In the fight against the recent COVID-19 pandemics, testing is crucial. Nasopharyngeal swabs and real-time RT-PCR are used for the detection of the viral RNA. The collection of saliva is non-invasive, pain-free and does not require trained personnel. An alternative to RT-PCR is loop-mediated isothermal amplification coupled with reverse transcription (RT-LAMP) that is easy to perform, quick and does not require a thermal cycler. The aim of this study was to test whether SARS-CoV-2 RNA can be detected directly in saliva using RT-LAMP. We have tested 16 primer mixes from the available literature in three rounds of sensitivity assays. The selected RT-LAMP primer mix has a limit of detection of 6 copies of viral RNA per reaction in comparison with RT-PCR with 1 copy per reaction. Whole saliva, as well as saliva collected using Salivette collection tubes, interfered with the RT-LAMP analysis. Neither Chelex-100 nor protease treatment of saliva prevented the inhibitory effect of saliva. With the addition of the ribonuclease inhibitor, the sensitivity of the RT-LAMP assay was 12 copies per reaction of RNA in Salivette\u00ae saliva samples and 6 copies per reaction of RNA in whole saliva samples. This study shows that it is possible to combine the use of saliva and RT-LAMP for SARS-CoV-2 RNA detection without RNA extraction which was confirmed on a small set of correctly diagnosed clinical samples. Further studies should prove whether this protocol is suitable for point of care testing in the clinical setting.","lamp_id":[{"id":"LPB00203","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00204","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00207","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00208","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00209","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":106,"pmid":16032744,"title":"Detection of respiratory syncytial virus genome by subgroups-A, B specific reverse transcription loop-mediated isothermal amplification (RT-LAMP)","year":2005,"journal":"Journal of Medical Virology","authors":"Masanobu Ushio, Ikuko Yui, Naoko Yoshida, Motoko Fujino, Toshihiro Yonekawa, Yoshinori Ota, Tsugunori Notomi, Tetsuo Nakayama","doi":"10.1002\/jmv.20424","country":"Japan","institute":"Tokyo Medical College","deparment":"Department of Pediatrics","abstract":"Annual seasonal outbreaks of respiratory syncytial virus (RSV) infection occur every winter. Most patients are diagnosed clinically by a rapid detection kit for RSV protein(s) from nasopharyngeal secretion (NPS), but some problems have been reported on the specificity and sensitivity of such rapid detection kits. To ratify these issues, a sensitive, specific, simple, and rapid molecular based diagnostic method is expected to be introduced and we have developed a method to detect the RSV genome of subgroups A and B independently by reverse transcription loop-mediated isothermal amplification (RT-LAMP). We detected the genomic RNA corresponding approximately to 0.1 TCID 50 in the sample by RT-LAMP for both RSV subgroups under isothermal condition within 60 min after extraction of RNA. Specific DNA amplification was monitored by a real-time turbidimeter and the quantity of RNA was calculated. The RSV genome was detected in 47 of 50 NPS by RT-LAMP, and in 42 by nested RT-PCR, whereas virus isolation was positive for 29 and enzyme-linked immunoassay (EIA) for 34. RSV subgroup A was detected in 25 by RSV RT-LAMP A, RSV subgroup B in 23 by RSV RT-LAMP B, and dual infection with RSV subgroups A and B was identified in one case. They were confirmed with digestion with a specific restriction enzyme, Bgl II. The results showed the potential clinical feasibility of RT-LAMP as a useful diagnostic tool for the detection of RSV with high sensitivity similar to nested RT-PCR.","lamp_id":[{"id":"LPB00205","pathogen":"HRSV A","target":"N"},{"id":"LPB00206","pathogen":"HRSV B","target":"N"}]},{"id":107,"pmid":33399137,"title":"Colorimetric loop-mediated isothermal amplification (LAMP) for cost-effective and quantitative detection of SARS-CoV-2: the change in color in LAMP-based assays quantitatively correlates with viral copy number","year":2020,"journal":"Analytical Methods","authors":"Everardo Gonz\u00e1lez-Gonz\u00e1lez, Itzel Montserrat Lara-Mayorga, Iram Pablo Rodr\u00edguez-S\u00e1nchez, Yu Shrike Zhang, Sergio O. Mart\u00ednez-Chapa, Grissel Trujillo-de Santiago, Mario Mois\u00e9s Alvarez","doi":"10.1039\/d0ay01658f","country":"Mexico","institute":"Tecnologico de Monterrey","deparment":"Centro de Biotecnolog\u00eda-FEMSA\/Departamento de Bioingenier\u00eda","abstract":"We demonstrate a loop-mediated isothermal amplification (LAMP) method to detect and amplify SARS-CoV-2 genetic sequences using a set of in-house designed initiators that target regions encoding the N protein. We were able to detect and amplify SARS-CoV-2 nucleic acids in the range of 62 to 2 \u00d7 10^5 DNA copies by this straightforward method. Using synthetic SARS-CoV-2 samples and RNA extracts from patients, we demonstrate that colorimetric LAMP is a quantitative method comparable in diagnostic performance to RT-qPCR (i.e., sensitivity of 92.85% and specificity of 81.25% in a set of 44 RNA extracts from patients analyzed in a hospital setting).","lamp_id":[{"id":"LPB00210","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00211","pathogen":"SARS-CoV-2","target":"N"}]},{"id":108,"pmid":23529294,"title":"Detection of Mycoplasma pneumoniae by colorimetric loop-mediated isothermal amplification","year":2013,"journal":"Acta Microbiologica et Immunologica Hungarica","authors":"Fei Zhao, Zhong Liu, Yixin Gu, Yuelian Yang, Di Xiao, Xiaoxia Tao, Fanliang Meng, Lihua He, Jianzhong Zhang","doi":"10.1556\/AMicr.60.2013.1.1","country":"","institute":"","deparment":"","abstract":"Mycoplasma pneumoniae (M. pneumoniae) is one of the most important pathogens that cause respiratory tract infection in children and adults. In this study, we describe a rapid and sensitive colorimetric loop mediated isothermal amplification (LAMP) method to detect M. pneumoniae. The specificity and sensitivity of this assay were detected with 21 common respiratory pathogens and 39 M. pneumoniae DNA. The sensitivity of LAMP was 100% among 39 M. pneumoniae isolates and the specificity was 100% among 9 members of other Mycoplasma and 12 common respiratory pathogens. The lowest detectable limit (LDL) of this assay was 10^2 copies, which detected by a series of standard M. pneumoniae DNA. To evaluate the clinical applicability of the LAMP assay, a total of 80 clinical samples were examined by conventional PCR, real-time PCR and the LAMP assays, respectively. The positive rates were 15.0%, 32.5% and 26.3%, respectively. This colorimetric LAMP assay demonstrated a high level of sensitivity comparable with that of conventional PCR for the detection of M. pneumoniae. It is a valuable method for simple, cost-effective and rapid detection of M. pneumoniae in the rural areas and basic clinical of China.","lamp_id":[{"id":"LPB00212","pathogen":"Mycoplasma pneumoniae","target":"P1"}]},{"id":109,"pmid":35260588,"title":"Portable real-time colorimetric LAMP-device for rapid quantitative detection of nucleic acids in crude samples","year":2022,"journal":"Scientific Reports","authors":"G Papadakis, A K Pantazis, N Fikas, S Chatziioannidou, V Tsiakalou, K Michaelidou, V Pogka, M Megariti, M Vardaki, K Giarentis, J Heaney, E Nastouli, T Karamitros, A Mentis, A Zafiropoulos, G Sourvinos, S Agelaki, E Gizeli","doi":"10.1038\/s41598-022-06632-7","country":"Greece","institute":"Foundation for Research and Technology-Hellas\/Science and Technology Park of Crete","deparment":"Institute of Molecular Biology and Biotechnology\/BIOPIX DNA TECHNOLOGY PC","abstract":"Loop-mediated isothermal amplification is known for its high sensitivity, specificity and tolerance to inhibiting-substances. In this work, we developed a device for performing real-time colorimetric LAMP combining the accuracy of lab-based quantitative analysis with the simplicity of point-of-care testing. The device innovation lies on the use of a plastic tube anchored vertically on a hot surface while the side walls are exposed to a mini camera able to take snapshots of the colour change in real time during LAMP amplification. Competitive features are the rapid analysis (< 30 min), quantification over 9 log-units, crude sample-compatibility (saliva, tissue, swabs), low detection limit (< 5 copies\/reaction), smartphone-operation, fast prototyping (3D-printing) and ability to select the dye of interest (Phenol red, HNB). The device's clinical utility is demonstrated in cancer mutations-analysis during the detection of 0.01% of BRAF-V600E-to-wild-type molecules from tissue samples and COVID-19 testing with 97% (Ct < 36.8) and 98% (Ct < 30) sensitivity when using extracted RNA and nasopharyngeal-swabs, respectively. The device high technology-readiness-level makes it a suitable platform for performing any colorimetric LAMP assay; moreover, its simple and inexpensive fabrication holds promise for fast deployment and application in global diagnostics.","lamp_id":[{"id":"LPB00213","pathogen":"SARS-CoV-2","target":"N"}]},{"id":110,"pmid":21177907,"title":"Development and evaluation of a reverse transcription-loop-mediated isothermal amplification assay for rapid detection of enterovirus 71","year":2011,"journal":"Journal of Clinical Microbiology","authors":"Tao Jiang, Juan Liu, Yong-Qiang Deng, Li-Juan Xu, Xiao-Feng Li, Jian-Feng Han, Rui-Yuan Cao, E-De Qin, Cheng-Feng Qin","doi":"10.1128\/JCM.02045-10","country":"China","institute":"Beijing Institute of Microbiology and Epidemiology","deparment":"State Key Laboratory of Pathogen and Biosecurity","abstract":"Human enterovirus 71 (EV71) is the major etiological agent of hand, foot, and mouth disease (HFMD), which is a common infectious disease in young children and infants. EV71 can cause various clinical manifestations and has been associated with severe neurological complications; it has resulted in fatalities during recent outbreaks in Asian-Pacific regions since 1997. The early and rapid detection is critical for prevention and control of EV71 infection, since no vaccine or antiviral drugs are currently available. In this study, a simple and sensitive reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid detection of EV71. The detection limit of the RT-LAMP assay was approximately 0.01 PFU per reaction mixture, and no cross-reactive amplification with other enteroviruses was observed. The assay was evaluated further with 40 clinical specimens and exhibited 92.9% sensitivity and 100% specificity. This RT-LAMP assay may become a useful alternative in clinical diagnosis of EV71, especially in resource-limited hospitals or rural clinics of China and other countries in the Asian-Pacific region.","lamp_id":[{"id":"LPB00215","pathogen":"EV-A71","target":"VP3"}]},{"id":111,"pmid":24747008,"title":"Development of a reverse transcription loop-mediated isothermal amplification assay for the rapid diagnosis of avian influenza A (H7N9) virus infection","year":2014,"journal":"Journal of Virological Methods","authors":"Mina Nakauchi, Ikuyo Takayama, Hitoshi Takahashi, Masato Tashiro, Tsutomu Kageyam","doi":"10.1016\/j.jviromet.2014.03.028","country":"Japan","institute":"National Institute of Infectious Diseases","deparment":"Influenza Virus Research Center","abstract":"A genetic diagnosis system for detecting avian influenza A (H7N9) virus infection using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technology was developed. The RT-LAMP assay showed no cross-reactivity with seasonal influenza A (H3N2 and H1N1pdm09) or influenza B viruses circulating in humans or with avian influenza A (H5N1) viruses. The sensitivity of the RT-LAMP assay was 42.47 copies\/reaction. Considering the high specificity and sensitivity of the assay for detecting the avian influenza A (H7N9) virus and that the reaction was completed within 30 min, the RT-LAMP assay developed in this study is a promising rapid diagnostic tool for avian influenza A (H7N9) virus infection.","lamp_id":[{"id":"LPB00216","pathogen":"Influenza A virus (H7N9)","target":"HA"}]},{"id":112,"pmid":33536475,"title":"Comparative evaluation of 19 reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2","year":2021,"journal":"Scientific Reports","authors":"Yajuan Dong, Xiuming Wu, Shenwei Li, Renfei Lu, Yingxue Li, Zhenzhou Wan, Jianru Qin, Guoying Yu, Xia Jin, Chiyu Zhang","doi":"10.1038\/s41598-020-80314-0","country":"China","institute":"Fudan University\/Henan Normal University\/Institut Pasteur of Shanghai\/Chinese Academy of Agricultural Sciences","deparment":"Shanghai Public Health Clinical Center\/College of Life Sciences\/Pathogen Discovery and Evolution Unit\/Tobacco Research Institute","abstract":"Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has caused a global pandemics. To facilitate the detection of SARS-CoV-2 infection, various RT-LAMP assays using 19 sets of primers had been developed, but never been compared. We performed comparative evaluation of the 19 sets of primers using 4 RNA standards and 29 clinical samples from COVID-19 patients. Six of 15 sets of primers were firstly identified to have faster amplification when tested with four RNA standards, and were further subjected to parallel comparison with the remaining four primer sets using 29 clinical samples. Among these 10 primer sets, Set-4 had the highest positive detection rate of SARS-CoV-2 (82.8%), followed by Set-10, Set-11, and Set-13 and Set-17 (75.9%). Set-14 showed the fastest amplification speed (Tt value < 8.5 min), followed by Set-17 (Tt value < 12.5 min). Based on the overall detection performance, Set-4, Set-10, Set-11, Set-13, Set-14 and Set-17 that target Nsp3, S, S, E, N and N gene regions of SARS-CoV-2, respectively, were determined to be better than the other primer sets. Two RT-LAMP assays with the Set-4 primers in combination with any one of four other primer sets (Set-14, Set-10, Set-11, and Set-13) were recommended to be used in the COVID-19 surveillance.","lamp_id":[{"id":"LPB00217","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"}]},{"id":113,"pmid":21767352,"title":"Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid detection of enterovirus 71","year":2011,"journal":"BMC Infectious Diseases","authors":"Weifeng Shi, Kun Li, Yun Ji, Qingbo Jiang, Mei Shi, Zuhuang Mi","doi":"10.1186\/1471-2334-11-197","country":"People's Republic of China","institute":"The Third Affiliated Hospital of Suzhou University","deparment":"Department of Clinical Laboratory","abstract":"Background: Hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) is very common in China. It is difficult to distinguish between EV71 and coxsackievirus A16 (CVA16) infections in clinical HFMD patients. Routine laboratory diagnosis of EV71 infection is time-consuming and requires expensive instruments. In this study, we have developed a one-step, single tube, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of EV71.\n\nMethods: Six primers that can recognize 6 distinct regions on the VP2 gene of EV71 were designed for RT-LAMP assay. The amplification was completed by incubating all reagents in a single tube with reverse transcriptase and Bst DNA polymerase under the isothermal condition (60\u00b0C) for 60 min, and could be evaluated by using GoldView staining under a handheld ultraviolet torch lamp or electrophoresis analysis.\n\nResults: A total of 123 specimens collected from suspicious patients with HFMD were simultaneously detected by RT-LAMP and PCR fluorescence probing assay. The RT-LAMP amplified products containing EV71 were digested by HinfI and TaqI restriction endonucleases; in contrast, non-specific products with CVA16, coxsackievirus A4 and coxsackievirus B3 could not be detected in RT-LAMP assay. Meanwhile, RT-LAMP assay could amplify EV71 virus with a detection limit of 1 PFU\/ml within 60 min. Compared with PCR fluorescence probing assay, RT-LAMP assay exhibited 98.4% identity during the detection of EV71 viral RNA without the missing of positive samples.\n\nConclusion: Our results indicated that RT-LAMP is a rapid, sensitive, specific and accurate method for the detection of EV71 in clinical specimens. Therefore, this developed method has potential application for rapid and comprehensive surveillance for EV71 infection, especially in developing country.","lamp_id":[{"id":"LPB00218","pathogen":"EV-A71","target":"VP2"}]},{"id":114,"pmid":35095200,"title":"An integrated microfluidic platform featuring real-time reverse transcription loop-mediated isothermal amplification for detection of COVID-19","year":2022,"journal":"Sensors and Actuators B: Chemical","authors":"You-Ru Jhou, Chih-Hung Wang, Huey-Pin Tsai, Yan-Shen Shan, Gwo-Bin Lee","doi":"10.1016\/j.snb.2022.131447","country":"Taiwan","institute":"National Tsing Hua University","deparment":"Department of Power Mechanical Engineering","abstract":"An integrated microfluidic platform (IMP) utilizing real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was developed here for detection and quantification of three genes of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; i.e., coronavirus diseases 2019 (COVID-19)): RNA-dependent RNA polymerase, the envelope gene, and the nucleocapsid gene for molecular diagnosis. The IMP comprised a microfluidic chip, a temperature control module, a fluidic control module that collectively carried out viral lysis, RNA extraction, RT-LAMP, and the real-time detection within 90 min in an automatic format. A limit of detection of 5 \u00d7 10^3 copies\/reaction for each gene was determined with three samples including synthesized RNAs, inactive viruses, and RNAs extracted from clinical samples; this compact platform could be a useful tool for COVID-19 diagnostics.","lamp_id":[{"id":"LPB00220","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00484","pathogen":"SARS-CoV-2","target":"RdRp"},{"id":"LPB00504","pathogen":"SARS-CoV-2","target":"E"}]},{"id":115,"pmid":33439160,"title":"Rapid molecular diagnostics of COVID-19 by RT-LAMP in a centrifugal polystyrene-toner based microdevice with end-point visual detection","year":2021,"journal":"Analyst","authors":"K\u00e9zia Gomes de Oliveira, Paulo Felipe Neves Estrela, Geovana de Melo Mendes, Carlos Abelardo dos Santos, Elis\u00e2ngela de Paula Silveira-Lacerda, Gabriela Rodrigues Mendes Duarte","doi":"10.1039\/d0an02066d","country":"Brazil","institute":"Universidade Federal de Goi\u00e1s","deparment":"Instituto de Qu\u00edmica","abstract":"Infection caused by the new coronavirus (SARS-CoV-2) has become a serious worldwide public health problem, and one of the most important strategies for its control is mass testing. Loop-mediated isothermal amplification (LAMP) has emerged as an important alternative to simplify the diagnostics of infectious diseases. In addition, an advantage of LAMP is that it allows for easy reading of the final result through visual detection. However, this step must be performed with caution to avoid contamination and false-positive results. LAMP performed on microfluidic platforms can minimize false-positive results, in addition to having potential for point-of-care applications. Here, we describe a polystyrene-toner (PS-T) centrifugal microfluidic device manually controlled by a fidget spinner for molecular diagnosis of COVID-19 by RT-LAMP, with integrated and automated colorimetric detection. The amplification was carried out in a microchamber with 5 \u03bcL capacity, and the reaction was thermally controlled with a thermoblock at 72 \u00b0C for 10 min. At the end of the incubation time, the detection of amplified RT-LAMP fragments was performed directly on the chip by automated visual detection. Our results demonstrate that it is possible to detect COVID-19 in reactions initiated with approximately 10-3 copies of SARS-CoV-2 RNA. Clinical samples were tested using our RT-LAMP protocol as well as by conventional RT-qPCR, demonstrating comparable performance to the CDC SARS-CoV-2 RT-qPCR assay. The methodology described in this study represents a simple, rapid, and accurate method for rapid molecular diagnostics of COVID-19 in a disposable microdevice, ideal for point-of-care testing (POCT) systems.","lamp_id":[{"id":"LPB00221","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":116,"pmid":34006857,"title":"A rapid, accurate, scalable, and portable testing system for COVID-19 diagnosis","year":2021,"journal":"Nature Communications","authors":"Guanhua Xun, Stephan Thomas Lane, Vassily Andrew Petrov, Brandon Elliott Pepa, Huimin Zhao","doi":"10.1038\/s41467-021-23185-x","country":"USA","institute":"University of Illinois at Urbana-Champaign","deparment":"Department of Bioengineering\/Carl R. Woese Institute for Genomic Biology","abstract":"The need for rapid, accurate, and scalable testing systems for COVID-19 diagnosis is clear and urgent. Here, we report a rapid Scalable and Portable Testing (SPOT) system consisting of a rapid, highly sensitive, and accurate assay and a battery-powered portable device for COVID-19 diagnosis. The SPOT assay comprises a one-pot reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) followed by PfAgo-based target sequence detection. It is capable of detecting the N gene and E gene in a multiplexed reaction with the limit of detection (LoD) of 0.44 copies\/\u03bcL and 1.09 copies\/\u03bcL, respectively, in SARS-CoV-2 virus-spiked saliva samples within 30 min. Moreover, the SPOT system is used to analyze 104 clinical saliva samples and identified 28\/30 (93.3% sensitivity) SARS-CoV-2 positive samples (100% sensitivity if LoD is considered) and 73\/74 (98.6% specificity) SARS-CoV-2 negative samples. This combination of speed, accuracy, sensitivity, and portability will enable high-volume, low-cost access to areas in need of urgent COVID-19 testing capabilities.","lamp_id":[{"id":"LPB00222","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00223","pathogen":"SARS-CoV-2","target":"E"}]},{"id":117,"pmid":33907239,"title":"Colorimetric RT-LAMP SARS-CoV-2 diagnostic sensitivity relies on color interpretation and viral load","year":2021,"journal":"Scientific Reports","authors":"Mateus N\u00f3brega Aoki, Bruna de Oliveira Coelho, Luiz Gustavo Bentim G\u00f3es, Paola Minoprio, Edison Luiz Durigon, Luis Gustavo Morello, Fabricio Klerynton Marchini, Irina Natassja Riediger, Maria do Carmo Debur, Helder I Nakaya, Lucas Blanes","doi":"10.1038\/s41598-021-88506-y","country":"Brazil","institute":"Carlos Chagas Institute","deparment":"Laboratory for Applied Science and Technology in Health","abstract":"The use of RT-LAMP (reverse transcriptase-loop mediated isothermal amplification) has been considered as a promising point-of-care method to diagnose COVID-19. In this manuscript we show that the RT-LAMP reaction has a sensitivity of only 200 RNA virus copies, with a color change from pink to yellow occurring in 100% of the 62 clinical samples tested positive by RT-qPCR. We also demonstrated that this reaction is 100% specific for SARS-CoV-2 after testing 57 clinical samples infected with dozens of different respiratory viruses and 74 individuals without any viral infection. Although the majority of manuscripts recently published using this technique describe only the presence of two-color states (pink = negative and yellow = positive), we verified by naked-eye and absorbance measurements that there is an evident third color cluster (orange), in general related to positive samples with low viral loads, but which cannot be defined as positive or negative by the naked eye. Orange colors should be repeated or tested by RT-qPCR to avoid a false diagnostic. RT-LAMP is therefore very reliable for samples with a RT-qPCR Ct < 30 being as sensitive and specific as a RT-qPCR test. All reactions were performed in 30 min at 65 \u00b0C. The use of reaction time longer than 30 min is also not recommended since nonspecific amplifications may cause false positives.","lamp_id":[{"id":"LPB00225","pathogen":"SARS-CoV-2","target":"N"}]},{"id":118,"pmid":25649029,"title":"Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for the Detection of Adenovirus 40 and 41","year":2015,"journal":"Food and Environmental Virology","authors":"P G Ziros, P A Kokkinos, A Allard, A Vantarakis","doi":"10.1007\/s12560-015-9182-8","country":"Greece","institute":"University of Patras","deparment":"Department of Public Health","abstract":"Human adenoviruses (hAdVs) of subgroup F (enteric serotypes 40 and 41) display characteristic gut tropism, in vivo, fastidious growth characteristics in cell culture, and are estimated to be associated with 5-20% worldwide of acute gastroenteritis cases among infants and young children. Adequate hAdV gastroenteritis case management requires laboratory-based diagnosis. The present study aimed to the development and evaluation of a simple and cost-effective, one-step, single-tube adenovirus type 40\/41 specific loop-mediated isothermal amplification (LAMP) assay for the detection of hAdV40\/41 DNA in environmental and\/or clinical samples, since no LAMP assay has previously been reported for the detection of these virus types. The assay targeted the hexon gene and had the advantages of being rapid, simple, specific, and sensitive. Results could be obtained within 60 min, under isothermal conditions at 69 \u00b0C. The detection limits for hAdV genomes were between 50 and 100 copies\/reaction for hAdV40 and hAdV41, and no cross-reactions with other selected viruses, were found. The assay was evaluated with clinical as well as environmental samples. The developed assay is expected to provide a potential molecular tool in obtaining greater knowledge of the hAdV40\/41 importance in the epidemiology and clinical manifestations of gastroenteritis.","lamp_id":[{"id":"LPB00226","pathogen":"HAdV","target":"Hexon"}]},{"id":119,"pmid":22155579,"title":"Detection of enterovirus 71 using reverse transcription loop-mediated isothermal amplification (RT-LAMP)","year":2012,"journal":"Journal of Virological Methods","authors":"Xiang Wang, Jun-ping Zhu, Qian Zhang, Zi-gang Xu, Fang Zhang, Zhi-hui Zhao, Wen-zhi Zheng, Li-shu Zheng","doi":"10.1016\/j.jviromet.2011.11.019","country":"China","institute":"National Institute for Viral Disease Control and Prevention","deparment":"State Key Laboratory for Molecular Virology and Genetic Engineering","abstract":"Reverse transcription loop-mediated isothermal amplification (RT-LAMP), which is a visual assay for nucleic acids, is performed in a single step using one tube at 65 \u00b0C for 1.5 h. In this study, RT-LAMP was established as a method for the detection of enterovirus 71 (EV71). The detection limit of the assay was approximately 10 copies, and no cross-reactivity was noted with Coxsackievirus A16, echovirus, human rotavirus (HRV) or norovirus. This assay, which offers greater sensitivity at a lower cost compared with the conventional reverse transcription polymerase chain reaction (RT-PCR), was validated using 252 clinical specimens that had been confirmed by laboratory diagnosis using RT-PCR. Both methods produced the same results with 52 positive samples. The RT-LAMP-based assay does not require specialised equipment, and therefore, it can be performed conveniently during an outbreak or under field conditions. In brief, the RT-LAMP-based assay provided a simple, rapid and efficient method for the detection of EV71 nucleic acid under field conditions.","lamp_id":[{"id":"LPB00228","pathogen":"EV-A71","target":"VP1"}]},{"id":120,"pmid":33903853,"title":"Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination","year":2021,"journal":"Heliyon","authors":"Shanshan Wu, Xiyang Liu, Shenglong Ye, Jianmin Liu, Wei Zheng, Xue Dong, Xiushan Yin","doi":"10.1016\/j.heliyon.2021.e06886","country":"China","institute":"Shenyang University of Chemical Technology","deparment":"College of Pharmaceutical and Biological Engineering","abstract":"RT-LAMP detection of SARS-CoV-2 has been demonstrated to be a valuable diagnostic method for the diagnosis of COVID-191,2, which can rapidly screen carriers of the virus to effectively control the spread of the SARS-CoV-2. Here, we present a combination of dyes for isothermal detection of SARS-CoV-2 as a commercial alternative, with expanded colorimetric spectrum. We compared them with commercial reagents and proved their suitability and sensitivity through clinical RNA samples. In addition, together with commercial single dye indicators, we believe the expanded color spectrum developed here as an indicator of rapid detection will promote the diagnosis of COVID-19.","lamp_id":[{"id":"LPB00229","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":121,"pmid":33975694,"title":"Integration of sample preparation with RNA-Amplification in a hand-held device for airborne virus detection","year":2021,"journal":"Analytica Chimica Acta","authors":"Xiao Jiang, Julia C Loeb, Maohua Pan, Trevor B Tilly, Arantza Eiguren-Fernandez, John A Lednicky, Chang-Yu Wu, Z Hugh Fan","doi":"10.1016\/j.aca.2021.338542","country":"","institute":"","deparment":"","abstract":"Aerosol transmission is one of the three major transmission routes of respiratory viruses. However, the dynamics and significance of the aerosol transmission route are not well understood, partially due to the lack of rapid and efficient tools for on-the-spot detection of airborne viruses. We report a hand-held device that integrates a 3D-printed sample preparation unit with a laminated paper-based RNA amplification unit. The sample preparation unit features an innovative reagent delivery scheme based on a ball-based valve capable of storing and delivering reagents through the rotation of the unit without manual pipetting, while the paper-based unit enables RNA enrichment and reverse transcription loop-mediated isothermal amplification (RT-LAMP). We have determined the detection limit of the integrated sample-preparation\/amplification device (SPAD) at 1 TCID50 H1N1 influenza viruses in 140 \u03bcL aqueous sample. Further, we integrated SPAD with a previously reported viable virus aerosol sampler (VIVAS), a water-vapor-based condensational growth system capable of collecting aerosolized virus particles (Pan et al., 2016) [1]. Using the combined VIVAS-SPAD platform, we have demonstrated the collection\/detection of lab-generated, airborne H1N1 influenza viruses in 65 min, suggesting that the platform has a potential for detecting and monitoring airborne virus transmission during outbreaks. The effective sampling and rapid detection of airborne viruses by the sample-to-answer platform will also help us better understand the dynamics and significance of aerosol transmission of infectious disease.","lamp_id":[{"id":"LPB00230","pathogen":"Influenza A virus (H1N1)","target":"HA"}]},{"id":122,"pmid":18572258,"title":"Development of reverse transcription loop-mediated isothermal amplification for rapid detection of H9 avian influenza virus","year":2008,"journal":"Journal of Virological Methods","authors":"Hao-tai Chen, Jie Zhang, De-hui Sun, Li-na Ma, Xiang-tao Liu, Xue-peng Cai, Yong-sheng Liu","doi":"10.1016\/j.jviromet.2008.05.009","country":"China","institute":"Lanzhou Veterinary Research Institute","deparment":"Key Laboratory of Animal Virology of Ministry of Agriculture","abstract":"Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a unique gene amplification method that can be completed within 45 min at 63 degrees C. In this study, RT-LAMP was used to develop a rapid and sensitive laboratory diagnostic system for the H9 subtype of avian influenza virus (AIV). The experiment results from the reference strains demonstrated that the established RT-LAMP sensitivity was 10-fold higher than that of RT-PCR, with the detection limit of 10 copies per reaction, and no cross-reactivity was observed from the samples of other related viruses including H5N1, H3N2 subtype of AIV and Newcastle disease virus. Furthermore, a total of 112 clinical samples were tested by RT-LAMP, RT-PCR, and virus isolation, respectively. All of the 85 positive specimens identified by virus isolation were also positive by RT-LAMP, while 7 of these samples were missed by RT-PCR. These results suggest that the present RT-LAMP system may provide a new avenue for the recognition of H9 subtype virus, and may be employed to screen for potential carriers in wild and domestic birds.","lamp_id":[{"id":"LPB00231","pathogen":"Influenza A virus (H9N2)","target":"HA"}]},{"id":123,"pmid":33535237,"title":"Rapid, Sensitive, and Specific Severe Acute Respiratory Syndrome Coronavirus 2 Detection: A Multicenter Comparison Between Standard Quantitative Reverse-Transcriptase Polymerase Chain Reaction and CRISPR-Based DETECTR","year":2020,"journal":"The Journal of Infectious Diseases","authors":"Eelke Brandsma, Han J M P Verhagen, Thijs J W van de Laar, Eric C J Claas, Marion Cornelissen, Emile van den Akker","doi":"10.1093\/infdis\/jiaa641","country":"The Netherlands","institute":"Sanquin Research\/University of Amsterdam","deparment":"Department of Hematopoiesis\/Landsteiner Laboratory","abstract":"Background: Recent advances in CRISPR-based diagnostics suggest that DETECTR, a combination of reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) and subsequent Cas12 bystander nuclease activation by amplicon-targeting ribonucleoprotein complexes, could be a faster and cheaper alternative to quantitative reverse-transcription polymerase chain reaction (qRT-PCR) without sacrificing sensitivity and\/or specificity.\n\nMethods: In this study, we compare DETECTR with qRT-PCR to diagnose coronavirus disease 2019 on 378 patient samples. Patient sample dilution assays suggest a higher analytical sensitivity of DETECTR compared with qRT-PCR; however, this was not confirmed in this large patient cohort, where we report 95% reproducibility between the 2 tests.\n\nResults: These data showed that both techniques are equally sensitive in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) providing additional value of DETECTR to the currently used qRT-PCR platforms. For DETECTR, different guide ribonucleic acids can be used simultaneously to obviate negative results due to mutations in N-gene. Lateral flow strips, suitable as a point-of-care test, showed a 100% correlation to the high-throughput DETECTR assay. More importantly, DETECTR was 100% specific for SARS-CoV-2 relative to other human coronaviruses.\n\nConclusions: Because there is no need for specialized equipment, DETECTR could be rapidly implemented as a complementary technically independent approach to qRT-PCR thereby increasing the testing capacity of medical microbiological laboratories and relieving the existent PCR platforms for routine non-SARS-CoV-2 diagnostic testing.","lamp_id":[{"id":"LPB00233","pathogen":"SARS-CoV-2","target":"N"}]},{"id":124,"pmid":33498408,"title":"A Novel Approach to the Bioluminescent Detection of the SARS-CoV-2 ORF1ab Gene by Coupling Isothermal RNA Reverse Transcription Amplification with a Digital PCR Approach","year":2021,"journal":"International Journal of Molecular Sciences","authors":"Zhongjie Fei, Rongbin Wei, Chu Cheng, Pengfeng Xiao","doi":"10.3390\/ijms22031017","country":"China","institute":"Southeast University","deparment":"State Key Laboratory of Bioelectronics","abstract":"The COVID-19 pandemic caused by the SARS-CoV-2 virus, which first emerged in December 2019, represents an ongoing global public health emergency. Here, we developed an improved and highly sensitive approach to SARS-CoV-2 detection via coupling bioluminescence in real-time (BART) and reverse-transcriptase loop-mediated amplification (RT-LAMP) protocols (RT-LAMP-BART) and was also compatible with a digital LAMP system (Rainsuit), which did not allow for real-time quantification but did, nonetheless, facilitate absolute quantification with a comparable detection limit of 10^4 copies\/mL. Through improving RNA availability in samples to ensure the target RNA present in reaction, we additionally developed a simulated digital RT-LAMP approach using this same principle to enlarge the overall reaction volume and to achieve real-time detection with a limit of detection of 10 copies\/mL, and with further improvements in the overall dynamic range of this assay system being achieved through additional optimization.","lamp_id":[{"id":"LPB00234","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":125,"pmid":null,"title":"A scalable saliva-based, extraction-free rt-lamp protocol for sars-cov-2 diagnosis","year":2020,"journal":"medRxiv","authors":"Paula Asprino, Fabiana Bettoni, Anamaria Camargo, Diego Coelho, Guilherme Coppini, Igor Correa, Erika Freitas, Lilian Inoue, Jo\u00e3o Paulo Kitajima, Mayra Kuroki, Cibele Masotti, Tatiana Marques, Alice Reis, Luiz Fernando Reis, Bibiana Santos, Ernande dos Santos, David Schlesinger, Cec\u00edlia Sena, Talita Spadaccini, Lucas Taniguti","doi":"10.1101\/2020.10.27.20220541","country":"Brazil","institute":"Hospital S\u00edrio-Liban\u00eas","deparment":"","abstract":"Scalable, cost-effective screening methods are an essential tool to control SARS-CoV-2 spread. We have developed a straight saliva-based, RNA extraction-free, RT-LAMP test that is comparable to current nasopharyngeal swab RT-PCR tests in both sensitivity and specificity. Using a 2-step readout of fluorescence and melting-point curve analysis, the test is scalable to more than 30,000 tests per day with average turnaround time of less than 3 hours. The test was validated using samples from 244 symptomatic patients, and showed sensitivity of 78.9% (vs. 85.5% for nasopharyngeal swabs RT-PCR) and specificity of 100% (vs. 100% for nasopharyngeal swabs RT-PCR). Our method is therefore accurate, robust, time and cost effective and therefore can be used for screening of SARS-CoV-2.","lamp_id":[{"id":"LPB00235","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"}]},{"id":126,"pmid":34795308,"title":"Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes","year":2021,"journal":"Scientific Reports","authors":"Daniel Urrutia-Cabrera, Roxanne Hsiang-Chi Liou, Jianxiong Chan, Sandy Shen-Chi Hung, Alex W Hewitt, Keith R Martin, Patrick Kwan, Thomas L Edwards, Raymond Ching-Bong Wong","doi":"10.1038\/s41598-021-01472-3","country":"Australia","institute":"Royal Victorian Eye and Ear Hospital\/University of Melbourne","deparment":"Centre for Eye Research Australia\/Department of Surgery","abstract":"The COVID-19 pandemic caused by SARS-CoV-2 has infected millions worldwide, therefore there is an urgent need to increase our diagnostic capacity to identify infected cases. Although RT-qPCR remains the gold standard for SARS-CoV-2 detection, this method requires specialised equipment in a diagnostic laboratory and has a long turn-around time to process the samples. To address this, several groups have recently reported the development of loop-mediated isothermal amplification (LAMP) as a simple, low cost and rapid method for SARS-CoV-2 detection. Herein we present a comparative analysis of three LAMP-based assays that target different regions of the SARS-CoV-2: ORF1ab RdRP, ORF1ab nsp3 and Gene N. We perform a detailed assessment of their sensitivity, kinetics and false positive rates for SARS-CoV-2 diagnostics in LAMP or RT-LAMP reactions, using colorimetric or fluorescent detection. Our results independently validate that all three assays can detect SARS-CoV-2 in 30 min, with robust accuracy at detecting as little as 1000 RNA copies and the results can be visualised simply by color changes. Incorporation of RT-LAMP with fluorescent detection further increases the detection sensitivity to as little as 100 RNA copies. We also note the shortcomings of some LAMP-based assays, including variable results with shorter reaction time or lower load of SARS-CoV-2, and false positive results in some experimental conditions and clinical saliva samples. Overall for RT-LAMP detection, the ORF1ab RdRP and ORF1ab nsp3 assays have faster kinetics for detection but varying degrees of false positives detection, whereas the Gene N assay exhibits no false positives in 30 min reaction time, which highlights the importance of optimal primer design to minimise false-positives in RT-LAMP. This study provides validation of the performance of LAMP-based assays as a rapid, highly sensitive detection method for SARS-CoV-2, which have important implications in development of point-of-care diagnostics for SARS-CoV-2.","lamp_id":[{"id":"LPB00236","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"},{"id":"LPB00237","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00238","pathogen":"SARS-CoV-2","target":"N"}]},{"id":127,"pmid":null,"title":"Head-to-head comparison of direct-input RT-PCR and RT-LAMP against RT-qPCR on extracted RNA for rapid SARS-CoV-2 diagnostics","year":2021,"journal":"medRxiv","authors":"Max J. Kellner, Martin Matl, James J. Ross, Jakob Schnabl, Dominik Handler, Robert Heinen, Justine Schaeffer, Peter Hufnagl, Alexander Indra, Marcus P.S. Dekens, Robert Fritsche-Polanz, Manuela F\u00f6dinger, Johannes Zuber, Vienna Covid-19 Detection Initiative (VCDI), Franz Allerberger, Andrea Pauli, Julius Brennecke","doi":"10.1101\/2021.01.19.21250079","country":"Austria","institute":"Vienna BioCenter (VBC)","deparment":"Research Institute of Molecular Pathology (IMP)\/Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA)","abstract":"Viral pandemics, such as Covid-19, pose serious threats to human societies. To control the spread of highly contagious viruses such as SARS-CoV-2, effective test-trace-isolate strategies require population-wide, systematic testing. Currently, RT-qPCR on extracted RNA is the only broadly accepted test for SARS-CoV-2 diagnostics, which bears the risk of supply chain bottlenecks, often exaggerated by dependencies on proprietary reagents. Here, we directly compare the performance of gold standard diagnostic RT-qPCR on extracted RNA to direct input RT-PCR, RT-LAMP and bead-LAMP on 384 primary patient samples collected from individuals with suspected Covid-19 infection. With a simple five minute crude sample inactivation step and one hour of total reaction time, we achieve assay sensitivities of 98% (direct RT-PCR), 93% (bead-LAMP) and 82% (RT-LAMP) for clinically relevant samples (diagnostic RT-qPCR Ct <35) and a specificity of >98%. For direct RT-PCR, our data further demonstrate a perfect agreement between real-time and end-point measurements, which allow a simple binary classification similar to the powerful visual readout of colorimetric LAMP assays. Our study provides highly sensitive and specific, easy to implement, rapid and cost-effective alternatives to diagnostic RT-qPCR tests.","lamp_id":[{"id":"LPB00239","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00240","pathogen":"SARS-CoV-2","target":"E"}]},{"id":128,"pmid":21142070,"title":"Predicting Viruses Accurately by a Novel Multiplex Microfluidic Loop-Mediated Isothermal Amplification Chip","year":2010,"journal":"Analytical Chemistry","authors":"Xueen Fang, Hui Chen, Shaoning Yu, Xingyu Jiang, Jilie Kong","doi":"10.1021\/ac102858j","country":"China","institute":"Fudan University\/National Center for Nanoscience and Technology","deparment":"Department of Chemistry and Institutes of Biomedical Sciences\/CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety","abstract":"Multiplex gene assay is a valuable molecular tool not only in academic science but also in clinical diagnostics. Multiplex PCR assays, DNA microarrays, and various nanotechnology-based methods are examples of major techniques developed for analyzing multiple genes; none of these, however, are suitable for point-of-care diagnostics, especially in resource-limited settings. In this report, we describe an octopus-like multiplex microfluidic loop-mediated isothermal amplification (m\u03bcLAMP) assay for the rapid analysis of multiple genes in the point-of-care format and provide a robust approach for predicting viruses. This assay with the ability of analyzing multiple genes qualitatively and quantitatively is highly specific, operationally simple, and cost\/time-effective with the detection limit of less than 10 copies\/\u03bcL in 2 \u03bcL quantities of sample within 0.5 h. We successfully developed a m\u03bcLAMP chip for differentiating three human influenza A substrains and identifying eight important swine viruses.","lamp_id":[{"id":"LPB00241","pathogen":"Influenza A virus (H3N6)","target":"M"},{"id":"LPB00242","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00243","pathogen":"Influenza A virus (H1N1)","target":"HA"}]},{"id":129,"pmid":33570399,"title":"Rapid One-pot Detection of SARS-CoV-2 based on Lateral Flow Assay in Clinical Samples","year":2021,"journal":"Analytical Chemistry","authors":"Chao Zhang, Tingting Zheng, Hua Wang, Wei Chen, Xiaoye Huang, Jianqi Liang, Liping Qiu, Da Han, Weihong Tan","doi":"10.1021\/acs.analchem.0c05059","country":"China","institute":"Institute of Molecular Medicine (IMM)\/Shanghai Jiao Tong University","deparment":"Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine\/State Key Laboratory of Oncogenes and Related Genes","abstract":"Rapid tests for pathogen identification and spread assessment are critical for infectious disease control and prevention. The control of viral outbreaks requires a nucleic acid diagnostic test that is sensitive and simple and delivers fast and reliable results. Here, we report a one-pot direct reverse transcript loop-mediated isothermal amplification (RT-LAMP) assay of SARS-CoV-2 based on a lateral flow assay in clinical samples. The entire contiguous sample-to-answer workflow takes less than 40 min from a clinical swab sample to a diagnostic result without professional instruments and technicians. The assay achieved an accuracy of 100% in 12 synthetic and 12 clinical samples compared to the data from PCR-based assays. We anticipate that our method will provide a universal platform for rapid and point-of-care detection of emerging infectious diseases.","lamp_id":[{"id":"LPB00244","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00245","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":130,"pmid":33779548,"title":"Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers","year":2021,"journal":"eLife","authors":"Qing Yang, Nicholas R Meyerson, Stephen K Clark, Camille L Paige, Will T Fattor, Alison R Gilchrist, Arturo Barbachano-Guerrero, Benjamin G Healy, Emma R Worden-Sapper, Sharon S Wu, Denise Muhlrad, Carolyn J Decker, Tassa K Saldi, Erika Lasda, Patrick Gonzales, Morgan R Fink, Kimngan L Tat, Cole R Hager, Jack C Davis, Christopher D Ozeroff, Gloria R Brisson, Matthew B McQueen, Leslie A Leinwand, Roy Parker, Sara L Sawyer","doi":"10.7554\/eLife.65113","country":"USA","institute":"University of Colorado Boulder\/Darwin Biosciences Inc","deparment":"BioFrontiers Institute\/Department of Molecular, Cellular,nand Developmental Biology","abstract":"Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification. The test has two steps: (1) heat saliva with a stabilization solution and (2) detect virus by incubating with a primer\/enzyme mix. After incubation, saliva samples containing the SARS-CoV-2 genome turn bright yellow. Because this test is pH dependent, it can react falsely to some naturally acidic saliva samples. We report unique saliva stabilization protocols that rendered 295 healthy saliva samples compatible with the test, producing zero false positives. We also evaluated the test on 278 saliva samples from individuals who were infected with SARS-CoV-2 but had no symptoms at the time of saliva collection, and from 54 matched pairs of saliva and anterior nasal samples from infected individuals. The Saliva TwoStep test described herein identified infections with 94% sensitivity and >99% specificity in individuals with sub-clinical (asymptomatic or pre-symptomatic) infections.","lamp_id":[{"id":"LPB00246","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00247","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00248","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":131,"pmid":32979593,"title":"Contamination-free visual detection of SARS-CoV-2 with CRISPR\/Cas12a: A promising method in the point-of-care detection","year":2020,"journal":"Biosensors and Bioelectronics","authors":"Yanju Chen, Ya Shi, Yin Chen, Zhangnv Yang, Hui Wu, Zhihui Zhou, Jue Li , Jianfeng Ping, Luping He, Hong Shen, Zhengxin Chen, Jian Wu, Yunsong Yu, Yanjun Zhang, Huan Chen","doi":"10.1016\/j.bios.2020.112642","country":"China","institute":"Zhejiang University","deparment":"College of Biosystems Engineering and Food Science","abstract":"The outbreaks of the infectious disease COVID-19 caused by SARS-CoV-2 seriously threatened the life of humans. A rapid, reliable and specific detection method was urgently needed. Herein, we reported a contamination-free visual detection method for SARS-CoV-2 with LAMP and CRISPR\/Cas12a technology. CRISPR\/Cas12a reagents were pre-added on the inner wall of the tube lid. After LAMP reaction, CRISPR\/Cas12a reagents were flowed into the tube and mixed with amplicon solution by hand shaking, which can effectively avoid possible amplicon formed aerosol contamination caused by re-opening the lid after amplification. CRISPR\/Cas12a can highly specific recognize target sequence and discriminately cleave single strand DNA probes (5'-6FAM 3'-BHQ1). With smart phone and portable 3D printing instrument, the produced fluorescence can be seen by naked eyes without any dedicated instruments, which is promising in the point-of-care detection. The whole amplification and detection process could be completed within 40 min with high sensitivity of 20 copies RNA of SARS-CoV-2. This reaction had high specificity and could avoid cross-reactivity with other common viruses such as influenza virus. For 7 positive and 3 negative respiratory swab samples provided by Zhejiang Provincial Center for Disease Control and Prevention, our detection results had 100% positive agreement and 100% negative agreement, which demonstrated the accuracy and application prospect of this method.","lamp_id":[{"id":"LPB00249","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00250","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00251","pathogen":"SARS-CoV-2","target":"N"}]},{"id":132,"pmid":33741959,"title":"An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing","year":2021,"journal":"Nature Communications","authors":"Kean Hean Ooi, Mengying Mandy Liu, Jie Wen Douglas Tay, Seok Yee Teo, Pornchai Kaewsapsak, Shengyang Jin, Chun Kiat Lee, Jingwen Hou, Sebastian Maurer-Stroh, Weisi Lin, Benedict Yan, Gabriel Yan, Yong-Gui Gao, Meng How Tan","doi":"10.1038\/s41467-021-21996-6","country":"Singapore","institute":"Nanyang Technological University\/Agency for Science Technology and Research","deparment":"School of Chemical and Biomedical Engineering\/Genome Institute of Singapore\/School of Biological Sciences","abstract":"Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 \u00b0C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.","lamp_id":[{"id":"LPB00252","pathogen":"SARS-CoV-2","target":"S"}]},{"id":133,"pmid":28322309,"title":"Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Cascade Invasive Reaction and Nanoparticle Hybridization for Subtyping of Influenza A Virus","year":2017,"journal":"Scientific Reports","authors":"Ying Chi, Yiyue Ge, Kangchen Zhao, Bingjie Zou, Bin Liu, Xian Qi, Qian Bian, Zhiyang Shi, Fengcai Zhu, Minghao Zhou, Lunbiao Cui, Chuan Su","doi":"10.1038\/srep44924","country":"China","institute":"Nanjing Medical University\/Jiangsu Provincial Center for Disease Prevention and Control","deparment":"Department of Pathogen Biology\/Institute of Pathogenic Microbiology","abstract":"Considering the fatal human victims and economic loss caused by influenza virus infection every year, methodologies for rapid and on-site detection of influenza viruses are urgently needed. LAMP is the most commonly used nucleic acid isothermal amplification technology suitable for on-site use. However, for multiplex LAMP, differentiation of the amplicons derived from multiple targets is still challengeable currently. Here we developed a multiplex RT-LAMP assay for simultaneous amplification of three prominent subtypes of influenza viruses (A\/H5, A\/H7 and 2009A\/H1). The amplicons were further identified by cascade invasive reaction and nanoparticle hybridization in separate target-specific detection tubes (referred to as mRT-LAMP-IRNH). The analytic sensitivities of the assay are 10 copies of RNA for all the three HA subtypes, and the specificity reached 100%. Clinical specimen analysis showed this assay had a combined sensitivity and specificity of 98.1% and 100%, respectively. Overall, the mRT-LAMP-IRNH assay can be used as a cost-saving method that utilizes a simple instrument to detect A\/H5, A\/H7, and 2009A\/H1 influenza viruses, especially in resource-limited settings.","lamp_id":[{"id":"LPB00253","pathogen":"Influenza A virus (H5N1)","target":"HA"},{"id":"LPB00254","pathogen":"Influenza A virus (H7N9)","target":"HA"},{"id":"LPB00255","pathogen":"Influenza A virus (H1N1)","target":"HA"}]},{"id":134,"pmid":25394781,"title":"Development of reverse-transcription loop-mediated isothermal amplification assay for rapid detection of novel avian influenza A (H7N9) virus","year":2014,"journal":"BMC Microbiology","authors":"Juan Liu, Qing-Gong Nian, Jing Li, Yi Hu, Xiao-Feng Li, Yu Zhang, Yong-Qiang Deng, Shun-Ya Zhu, Qing-Yu Zhu, E-De Qin, Tao Jiang, Cheng-Feng Qin","doi":"10.1186\/s12866-014-0271-x","country":"China","institute":"Beijing Institute of Microbiology and Epidemiology\/PLA Air Force General Hospital","deparment":"Department of Virology\/Department of Transfusion Medicine","abstract":"Background: The emerged human infection with avian influenza A (H7N9) virus in China since 2013 has aroused global concerns. There is great demand for simple and rapid diagnostic method for early detection of H7N9 to provide timely treatment and disease control. The aim of the current study was to develop a rapid, accurate and feasible reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of H7N9 virus.\n\nResults: The detection limits of the H7- and N9-specific RT-LAMP assay were both approximately 0.2 PFU per reaction. No cross-reactivity was observed with other subtype of influenza viruses or common respiratory viral pathogens. The assay worked well with clinical specimens from patients and chickens, and exhibited high specificity and sensitivity.\n\nConclusions: The H7\/N9 specific RT-LAMP assay was sensitive and accurate, which could be a useful alternative in clinical diagnostics of influenza A (H7N9) virus, especially in the hospitals and laboratories without sophisticated diagnostic systems.","lamp_id":[{"id":"LPB00256","pathogen":"Influenza A virus (H7N9)","target":"HA"},{"id":"LPB00257","pathogen":"Influenza A virus (H7N9)","target":"NA"}]},{"id":135,"pmid":23936359,"title":"Rapid and Sensitive Detection of Novel Avian-Origin Influenza A (H7N9) Virus by Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Lateral-Flow Device","year":2013,"journal":"PLoS One","authors":"Yiyue Ge, Bin Wu, Xian Qi, Kangchen Zhao, Xiling Guo, Yefei Zhu, Yuhua Qi, Zhiyang Shi, Minghao Zhou, Hua Wang, Lunbiao Cui","doi":"10.1371\/journal.pone.0069941","country":"China","institute":"Jiangsu Provincial Center for Disease Control and Prevention","deparment":"Institute of Pathogenic Microbiology","abstract":"A severe disease in humans caused by a novel avian-origin influenza A (H7N9) virus emerged in China recently, which has caused at least 128 cases and 26 deaths. Rapid detection of the novel H7N9 virus is urgently needed to differentiate the disease from other infections, and to facilitate infection control as well as epidemiologic investigations. In this study, a reverse transcription loop-mediated isothermal amplification combined with a lateral flow device (RT-LAMP-LFD) assay to rapidly detect H7N9 virus was developed and evaluated. The RT-LAMP primers were designed to target the haemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus. Results of 10-fold dilution series assays showed that analysis of RT-LAMP products by the LFD method was as sensitive as real-time turbidity detection, and that the analytic sensitivities of the HA and NA RT-LAMP assays were both 10 copies of synthetic RNA. Furthermore, both the assays showed 100% clinical specificity for identification of H7N9 virus. The performance characteristics of the RT-LAMP-LFD assay were evaluated with 80 clinical specimens collected from suspected H7N9 patients. The NA RT-LAMP-LFD assay was more sensitive than real time RT-PCR assay. Compared with a combination of virus culture and real-time RT-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the RT-LAMP-LFD assay were all 100%. Overall, The RT-LAMP-LFD assay established in this study can be used as a reliable method for early diagnosis of the avian-origin influenza A (H7N9) virus infection.","lamp_id":[{"id":"LPB00258","pathogen":"Influenza A virus (H7N9)","target":"HA"},{"id":"LPB00259","pathogen":"Influenza A virus (H7N9)","target":"NA"}]},{"id":136,"pmid":24006004,"title":"Rapid and Sensitive Detection of H7N9 Avian Influenza Virus by Use of Reverse Transcription\u2013Loop-Mediated Isothermal Amplification","year":2013,"journal":"Journal of Clinical Microbiology","authors":"Jinhai Zhang, Youjun Feng, Dan Hu, Heng Lv, Jing Zhu, Min Cao, Feng Zheng, Jin Zhu, Xiufang Gong, Lina Hao, Swaminath Srinivas, Hao Ren, Zhongtian Qi, Bingjun Li, Changjun Wang","doi":"10.1128\/JCM.01907-13","country":"China","institute":"Research Institute for Medicine of Nanjing Command","deparment":"Department of Epidemiology","abstract":"An epidemic of human H7N9 influenza virus infection recently emerged in China whose clinical features include high mortality and which has also resulted in serious economic loss. The novel reassortant avian-origin influenza A (H7N9) virus which was the causative agent of this epidemic raised the possibility of triggering a large-scale influenza pandemic worldwide. It seemed likely that fast molecular detection assays specific for this virus would be in great demand. Here, we report a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of the hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus, the minimum detection limit of which was evaluated using in vitro RNA transcription templates. In total, 135 samples from clinical specimens (from either patients or poultry) were tested using this method in comparison with the real-time PCR recommended by the World Health Organization (WHO). Our results showed that (i) RT-LAMP-based trials can be completed in approximately 12 to 23 min and (ii) the detection limit for the H7 gene is around 10 copies per reaction, similar to that of the real-time PCR, whereas the detection limit for its counterpart the N9 gene is 5 copies per reaction, a 100-fold-higher sensitivity than the WHO-recommended method. Indeed, this excellent performance of our method was also validated by the results for a series of clinical specimens. Therefore, we believe that the simple, fast, and sensitive method of RT-LAMP might be widely applied for detection of H7N9 infections and may play a role in prevention of an influenza pandemic.","lamp_id":[{"id":"LPB00260","pathogen":"Influenza A virus (H7N9)","target":"HA"},{"id":"LPB00261","pathogen":"Influenza A virus (H7N9)","target":"NA"}]},{"id":137,"pmid":33412756,"title":"Development of reverse transcription loop-mediated isothermal amplification assays for point-of-care testing of avian influenza virus subtype H5 and H9","year":2020,"journal":"Genomics & Informatics","authors":"Songzi Zhang, Juyoun Shin, Sun Shin, Yeun-Jun Chung","doi":"10.5808\/GI.2020.18.4.e40","country":"Korea","institute":"The Catholic University of Korea","deparment":"Department of Biomedicine & Health Sciences\/Department of Microbiology\/Precision Medicine Research Center\/Integrated Research Center for Genome Polymorphism","abstract":"Avian influenza (AIV) outbreaks can induce fatal human pulmonary infections in addition to economic losses to the poultry industry. In this study, we aimed to develop a rapid and sensitive point-of-care AIV test using loop-mediated isothermal amplification (LAMP) technology. We designed three sets of reverse transcription LAMP (RT-LAMP) primers targeting the matrix (M) and hemagglutinin (HA) genes of the H5 and H9 subtypes. RT-LAMP targeting the universal M gene was designed to screen for the presence of AIV and RT-LAMP assays targeting H5-HA and H9-HA were designed to discriminate between the H5 and H9 subtypes. All three RT-LAMP assays showed specific amplification results without nonspecific reactions. In terms of sensitivity, the detection limits of our RT-LAMP assays were 100 to 1,000 RNA copies per reaction, which were 10 times more sensitive than the detection limits of the reference reverse\u2012transcription polymerase chain reaction (RT-PCR) (1,000 to 10,000 RNA copies per reaction). The reaction time of our RT-LAMP assays was less than 30 minutes, which was approximately four times quicker than that of conventional RT-PCR. Altogether, these assays successfully detected the existence of AIV and discriminated between the H5 or H9 subtypes with higher sensitivity and less time than the conventional RT-PCR assay.","lamp_id":[{"id":"LPB00262","pathogen":"Influenza A virus (H5)","target":"M"},{"id":"LPB00263","pathogen":"Influenza A virus (H5)","target":"HA"},{"id":"LPB00264","pathogen":"Influenza A virus (H9)","target":"HA"}]},{"id":138,"pmid":24689044,"title":"Development of a Reverse Transcription Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Subtype H7N9 Avian Influenza Virus","year":2014,"journal":"BioMed Research International","authors":"Hongmei Bao, Yuhui Zhao, Yunhe Wang, Xiaolong Xu, Jianzhong Shi, Xianying Zeng, Xiurong Wang, Hualan Chen","doi":"10.1155\/2014\/525064","country":"China","institute":"Harbin Veterinary Research Institute","deparment":"Animal Influenza Laboratory of the Ministry of Agriculture","abstract":"A novel influenza A (H7N9) virus has emerged in China. To rapidly detect this virus from clinical samples, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7N9 virus. The minimum detection limit of the RT-LAMP assay was 0.01 PFU H7N9 virus, making this method 100-fold more sensitive to the detection of the H7N9 virus than conventional RT-PCR. The H7N9 virus RT-LAMP assays can efficiently detect different sources of H7N9 influenza virus RNA (from chickens, pigeons, the environment, and humans). No cross-reactive amplification with the RNA of other subtype influenza viruses or of other avian respiratory viruses was observed. The assays can effectively detect H7N9 influenza virus RNA in drinking water, soil, cloacal swab, and tracheal swab samples that were collected from live poultry markets, as well as human H7N9 virus, in less than 30 min. These results suggest that the H7N9 virus RT-LAMP assays were efficient, practical, and rapid diagnostic methods for the epidemiological surveillance and diagnosis of influenza A (H7N9) virus from different resource samples.","lamp_id":[{"id":"LPB00265","pathogen":"Influenza A virus (H7N9)","target":"NA"}]},{"id":139,"pmid":22028606,"title":"An Updated Loop-Mediated Isothermal Amplification Method for Rapid Diagnosis of H5N1 Avian Influenza Viruses","year":2011,"journal":"Tropical Medicine and Health","authors":"Duc Tuan Dinh, Mai Thi Quynh Le, Cuong Duc Vuong, Futoshi Hasebe, Kouichi Morita","doi":"10.2149\/tmh.2010-21","country":"Japan","institute":"Institute of Tropical Medicine","deparment":"Department of Virology","abstract":"We designed a new set of primers for reverse transcriptase loop-mediated isothermal amplification (RTLAMP) to specifically amplify the HA gene of avian influenza viruses subtype H5N1. By testing nine H5N1 virus strains and 41 clinical samples collected in Northern Vietnam, we found that the new primers showed higher sensitivity and specificity than the previously published RT-LAMP primers and were comparable to the RT-PCR method currently recommended by WHO. These results suggest that our RT-LAMP assay may be a better choice as a diagnostic tool for current H5N1 influenza virus infection.","lamp_id":[{"id":"LPB00266","pathogen":"Influenza A virus (H5N1)","target":"HA"}]},{"id":140,"pmid":20032626,"title":"Development of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for detection of avian influenza viruses in field specimens","year":2010,"journal":"Journal of Veterinary Medical Science","authors":"Sakar Shivakoti, Hiroshi Ito, Toshiyuki Murase, Etsuro Ono, Hiroki Takakuwa, Tetsu Yamashiro, Koichi Otsuki, Toshihiro Ito","doi":"10.1292\/jvms.09-0473","country":"Japan","institute":"Tottori University","deparment":"Laboratory of Veterinary Public Health","abstract":"Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an established gene amplification method for rapid diagnosis of various infectious diseases. In order to detect avian influenza viruses, particularly in field specimens, specific primers targeting the matrix gene were designed. Thirty-four virus samples, including isolates from wild and domestic avian hosts belonging to various geographical areas, were used to confirm the validity of the primers. All samples were confirmed to be positive in less than 1 hr. The RT-LAMP assay was also able to detect avian influenza virus in the various field samples, such as swabs, tissues, and feces. These results indicate that the developed RT-LAMP assay with uniquely designed primers is potentially useful in comprehensive avian influenza surveillance.","lamp_id":[{"id":"LPB00267","pathogen":"Influenza A virus (H5N1)","target":"M"}]},{"id":141,"pmid":17218021,"title":"Rapid diagnosis of H5N1 avian influenza virus infection by newly developed influenza H5 hemagglutinin gene-specific loop-mediated isothermal amplification method","year":2007,"journal":"Journal of Virological Methods","authors":"Masaki Imai, Ai Ninomiya, Harumi Minekawa, Tsugunori Notomi, Toru Ishizaki, Phan Van Tu, Nguyen Thi Kim Tien, Masato Tashiro, Takato Odagiri","doi":"10.1016\/j.jviromet.2006.12.004","country":"Japan","institute":"National Institute of Infectious Diseases","deparment":"Department of Virology 3","abstract":"Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a unique gene amplification method that can be completed within 35 min at 62.5 degrees C. In the present study, RT-LAMP was used to develop a rapid and sensitive laboratory diagnostic system for the H5N1 highly pathogenic avian influenza (HPAI). The sensitivity of the system was 0.1-0.01 plaque-forming units per reaction for HPAI-H5N1 viruses belonging to the genetically and antigenically distinct clade 1, represented by A\/Vietnam\/JP1203\/2004, and clade 2, represented by A\/Indonesia\/JP283\/2006. This RT-LAMP sensitivity is 10-fold higher than the sensitivity of standard one-step RT-PCR. By using viral RNAs extracted from avian influenza viruses of H1-H15 hemagglutinin (HA) subtypes and human pathogenic respiratory viruses, it was confirmed that the RT-LAMP system amplifies specifically RNA of the H5 subtype virus. The system detected H5-HA genes in throat swabs collected from humans as well as from wild birds. These results suggest that the present RT-LAMP system is a useful diagnostic tool for surveillance of recent outbreaks of the HPAI-H5N1 virus.","lamp_id":[{"id":"LPB00268","pathogen":"Influenza A virus (H5N1)","target":"HA"}]},{"id":142,"pmid":21930158,"title":"Development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of avian influenza virus subtype H7","year":2012,"journal":"Journal of Virological Methods","authors":"Hongmei Bao, Xiurong Wang, Yuhui Zhao, Xiaodong Sun, Yanbing Li, Yongzhong Xiong, Hualan Chen","doi":"10.1016\/j.jviromet.2011.08.023","country":"China","institute":"Harbin Veterinary Research Institute","deparment":"Animal Influenza Laboratory of the Ministry of Agriculture","abstract":"A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7 avian influenza virus (H7 AIV) isotype was developed. The minimum detection limit of the RT-LAMP assay was 0.1-0.01 PFU per reaction for H7 AIV RNA, making this assay 100-fold more sensitive than the conventional RT-PCR method. This RT-LAMP assay also has the capacity to detect both high- and low-pathogenic H7 AIV strains. Using a pool of RNAs extracted from influenza viruses corresponding to all 15 HA subtypes (in addition to other avian pathogenic viruses), the RT-LAMP system was confirmed to amplify only H7 AIV RNA. Furthermore, specific pathogen free (SPF) chickens were infected artificially with H7 AIV, throat and cloacal swabs were collected, and viral shedding was examined using viral isolation, RT-PCR and RT-LAMP. Shedding was detected following viral isolation and RT-LAMP one day after infection, whereas viral detection using RT-PCR was effective only on day 3 post-infection. These results indicate that the RT-LAMP method could facilitate epidemiological surveillance and the rapid diagnosis of the avian influenza subtype H7.","lamp_id":[{"id":"LPB00269","pathogen":"Influenza A virus (H7N1)","target":"HA"}]},{"id":143,"pmid":33657176,"title":"Development of a multiplex Loop-Mediated Isothermal Amplification (LAMP) assay for on-site diagnosis of SARS CoV-2","year":2021,"journal":"PLoS One","authors":"Woong Sik Jang, Da Hye Lim, Jung Yoon, Ahran Kim, Minsup Lim, Jeonghun Nam, Richard Yanagihara, Sook-Won Ryu, Bo Kyeung Jung, Nam-Hee Ryoo, Chae Seung Lim","doi":"10.1371\/journal.pone.0248042","country":"Republic of Korea","institute":"Korea University Guro Hospital","deparment":"College of Medicine","abstract":"A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP\/E, RdRP\/N, E\/N, RdRP\/E\/N and RdRP\/N\/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP\/E and RdRP\/N\/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex\u2122 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek\u2122 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)\/N (CY5)\/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP\/N: 96.92%) to that of the Allplex\u2122 2019-nCoV Assay (100%) and superior to those of PowerChek\u2122 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP\/E: 95.38%).","lamp_id":[{"id":"LPB00270","pathogen":"SARS-CoV-2","target":"RdRp"},{"id":"LPB00271","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00272","pathogen":"SARS-CoV-2","target":"N"}]},{"id":144,"pmid":33535813,"title":"Development of multiplexed reverse-transcription loop-mediated isothermal amplification for detection of SARS-CoV-2 and influenza viral RNA","year":2021,"journal":"Biotechniques","authors":"Yinhua Zhang, Nathan A Tanner","doi":"10.2144\/btn-2020-0157","country":"USA","institute":"New England Biolabs","deparment":"Nucleic Acid Replication Division","abstract":"The ongoing pandemic has demonstrated the utility of widespread surveillance and diagnostic detection of the novel SARS-CoV-2. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) has enabled broader testing, but current LAMP tests only detect single targets and require separate reactions for controls. With flu season in the Northern Hemisphere, the ability to screen for multiple targets will be increasingly important, and the ability to include internal controls in RT-LAMP allows for improved efficiency. Here we describe multiplexed RT-LAMP with four targets (SARS-CoV-2, influenza A, influenza B, human RNA) in a single reaction using real-time and end point fluorescence detection. Such increased functionality of RT-LAMP will enable even broader adoption of this molecular testing approach and aid in the fight against this public health threat.","lamp_id":[{"id":"LPB00273","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00274","pathogen":"Influenza A virus (H3N2)","target":"M"},{"id":"LPB00275","pathogen":"Influenza B virus","target":"NS"},{"id":"LPB00276","pathogen":"SARS-CoV-2","target":"N"}]},{"id":145,"pmid":33736798,"title":"A novel One-pot rapid diagnostic technology for COVID-19","year":2021,"journal":"Analytica Chimica Acta","authors":"Junmin Li, Xuejiao Hu, Xiaoming Wang, Jianing Yang, Lei Zhang, Qianyun Deng, Xiqin Zhang, Zixia Wang, Tieying Hou, Shan Li","doi":"10.1016\/j.aca.2021.338310","country":"People's Republic of China","institute":"South China University of Technology","deparment":"MOE International Joint Lab for Synthetic Biology and Medicines","abstract":"Novel coronavirus disease (COVID-19) caused by SARS-CoV-2 is an ongoing global pandemic associated with high rates of morbidity and mortality. RT-qPCR has become the diagnostic standard for the testing of SARS-CoV-2 in most countries. COVID-19 diagnosis generally relies upon RT-qPCR-mediated identification of SARS-CoV-2 viral RNA, which is costly, labor-extensive, and requires specialized training and equipment. Herein, we established a novel one-tube rapid diagnostic approach based upon formamide and colorimetric RT-LAMP (One-Pot RT-LAMP) that can be used to diagnose COVID-19 without the extraction of specific viral RNA. The technique could visually detect SARS-CoV-2 within 45 min with a limit of detection of 5 copies per reaction in extracted RNA, and about 7.66 virus copies per \u03bcL in viral transport medium. The One-Pot RT-LAMP test showed a high specificity without cross-reactivity with 12 viruses including SARS-CoV, MERS-CoV, and human infectious influenza virus (H1N1\/H3N2 of influenza A and B virus, ect. We validated this One-Pot RT-LAMP approach by its successful use for the analysis of 45 clinical nasopharyngeal swab samples, yielding results identical to those of traditional RT-qPCR analyses, while achieving good selectivity and sensitivity relative to a commercial RT-qPCR approach. As such, this One-Pot RT-LAMP technology may be a valid means of conducting high-sensitivity, low-cost and rapid SARS-CoV-2 identification without the extraction of viral RNA.","lamp_id":[{"id":"LPB00277","pathogen":"SARS-CoV-2","target":"N"}]},{"id":146,"pmid":33649735,"title":"Handheld Point-of-Care System for Rapid Detection of SARS-CoV-2 Extracted RNA in under 20 min","year":2021,"journal":"ACS Central Science","authors":"Jesus Rodriguez-Manzano, Kenny Malpartida-Cardenas, Nicolas Moser, Ivana Pennisi, Matthew Cavuto, Luca Miglietta, Ahmad Moniri, Rebecca Penn, Giovanni Satta, Paul Randell, Frances Davies, Frances Bolt, Wendy Barclay, Alison Holmes, Pantelis Georgiou","doi":"10.1021\/acscentsci.0c01288","country":"United Kingdom","institute":"Imperial College London","deparment":"Department of Infectious Disease\/Department of Electrical and Electronic Engineering","abstract":"The COVID-19 pandemic is a global health emergency characterized by the high rate of transmission and ongoing increase of cases globally. Rapid point-of-care (PoC) diagnostics to detect the causative virus, SARS-CoV-2, are urgently needed to identify and isolate patients, contain its spread and guide clinical management. In this work, we report the development of a rapid PoC diagnostic test (<20 min) based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and semiconductor technology for the detection of SARS-CoV-2 from extracted RNA samples. The developed LAMP assay was tested on a real-time benchtop instrument (RT-qLAMP) showing a lower limit of detection of 10 RNA copies per reaction. It was validated against extracted RNA from 183 clinical samples including 127 positive samples (screened by the CDC RT-qPCR assay). Results showed 91% sensitivity and 100% specificity when compared to RT-qPCR and average positive detection times of 15.45 \u00b1 4.43 min. For validating the incorporation of the RT-LAMP assay onto our PoC platform (RT-eLAMP), a subset of samples was tested (n = 52), showing average detection times of 12.68 \u00b1 2.56 min for positive samples (n = 34), demonstrating a comparable performance to a benchtop commercial instrument. Paired with a smartphone for results visualization and geolocalization, this portable diagnostic platform with secure cloud connectivity will enable real-time case identification and epidemiological surveillance.","lamp_id":[{"id":"LPB00279","pathogen":"SARS-CoV-2","target":"N"}]},{"id":147,"pmid":33681254,"title":"SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12","year":2021,"journal":"Frontiers in Medicine","authors":"Alfredo Garcia-Venzor, Bertha Rueda-Zarazua, Eduardo Marquez-Garcia, Vilma Maldonado, Angelica Moncada-Morales, Hiram Olivera, Irma Lopez, Joaquin Zu\u00f1iga, Jorge Melendez-Zajgla","doi":"10.3389\/fmed.2021.627679","country":"Mexico","institute":"Instituto Nacional de Medicina Gen\u00f3mica","deparment":"Cancer Functional Genomics Laboratory","abstract":"As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies\/\u03bcL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.","lamp_id":[{"id":"LPB00280","pathogen":"SARS-CoV-2","target":"N"}]},{"id":148,"pmid":30351335,"title":"Rapid detection of multiple respiratory viruses based on microfluidic isothermal amplification and a real-time colorimetric method","year":2018,"journal":"Lab on a Chip","authors":"Ruili Wang, Rongtao Zhao, Yang Li, Wen Kong, Xudong Guo, Yi Yang, Feng Wu, Wanying Liu, Hongbin Song, Rongzhang Hao","doi":"10.1039\/c8lc00841h","country":"China","institute":"Institute of Disease Control and Prevention\/Institute of Chemistry\/University of Chinese Academy of Sciences","deparment":"Beijing National Laboratory for Molecular Sciences (BNLMS)","abstract":"Respiratory viruses are major threats causing development of acute respiratory tract infections, which are common causes of illness and death throughout the world. Here, an integrated microsystem based on real-time colorimetry was developed for diagnosing multiple respiratory viruses. The microsystem employed magnetic beads for nucleic acid extraction and an eight-channel microfluidic array chip integrated with a loop-mediated isothermal amplification system for point-of-care screening of respiratory viruses. The overall detection process (including sample collection, nucleic acid extraction, sample loading, real-time detection, and signal output) could be completed within 1 h. Our results show that the developed method could specifically recognize influenza A virus subtypes (H1N1, H3N2, H5N1, and H7N9), influenza B virus, and human adenoviruses. The results obtained with 109 clinical samples indicate that the developed method has high specificity (100%, confidence interval 94.9-100.0) and sensitivity (96%, confidence interval 78.1-99.9). The integration of magnetic bead-based pre-treatment techniques and microfluidic isothermal amplification provides an effective solution for rapidly detecting etiological agents of respiratory diseases. The strategy of using a closed chip system and real-time colorimetry reduced aerosol contamination and ensured the accuracy of the results. The developed method provides an effective alternative for rapid point-of-care screening for viruses that cause respiratory disease syndromes and further aids in accurate and timely detection to control and prevent the spread of respiratory diseases caused by such pathogens.","lamp_id":[{"id":"LPB00281","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00282","pathogen":"Influenza A virus (H3N2)","target":"HA"},{"id":"LPB00283","pathogen":"Influenza A virus (H5N1)","target":"HA"},{"id":"LPB00284","pathogen":"Influenza A virus (H7N9)","target":"HA"},{"id":"LPB00285","pathogen":"Influenza B virus","target":"HA"},{"id":"LPB00286","pathogen":"HAdV","target":"Hexon"}]},{"id":149,"pmid":30735248,"title":"Development of real-time fluorescent reverse transcription loop-mediated isothermal amplification assays for rhinovirus detection","year":2019,"journal":"Journal of Medical Virology","authors":"Mina Nakauchi, Ikuyo Takayama, Hitoshi Takahashi, Shohei Semba, Shinji Saito,  Hideyuki Kubo,  Atsushi Kaida,  Kunihiro Oba, Shiho Nagata, Takato Odagiri, Tsutomu Kageyama","doi":"10.1002\/jmv.25427","country":"Japan","institute":"National Institute of Infectious Diseases","deparment":"Influenza Virus Research Center","abstract":"Human rhinoviruses (RVs) belong to the genus Enterovirus of the family Picornaviridae, and are classified into RV-A, -B, and -C species. Two assays were developed to detect RVs by a real-time fluorescent reverse transcription loop-mediated isothermal amplification method: one was designed based on the 5\u2032-untranslated regions (UTRs) of RV-A and -B, and the other was designed based on the 5\u2032-UTR of RV-C. The competence of both assays for the diagnosis of RV infection was tested using isolated viruses and compared with real-time reverse transcription polymerase chain reaction assays on clinical specimens. Neither assay demonstrated cross-reactivity with other tested enteroviruses, and they detected 19 out of 21 tested RV-As and seven out of eight tested RV-Cs. The specificity of the assays was 100% for the detection of RVs and their sensitivity for RV-A and RV-C was 86.3% and 77.3%, respectively, on clinical specimens by the combined use of both assays. Considering that both developed assays were highly specific and detected the majority of recently circulating RVs, they are helpful for the diagnosis of RV infection. Consequently, the results generated by these assays will enhance the surveillance of respiratory illness and the study of the roles of RVs associated with clinical features and disease severity.","lamp_id":[{"id":"LPB00287","pathogen":"HRV","target":"5\u2032\u2010UTR"}]},{"id":150,"pmid":28105842,"title":"Absolute Quantification of H5-Subtype Avian Influenza Viruses Using Droplet Digital Loop-Mediated Isothermal Amplification","year":2017,"journal":"Analytical Chemistry","authors":"Yi Hu, Peng Xu, Jing Luo, Hongxuan He, Wenbin Du","doi":"10.1021\/acs.analchem.6b03328","country":"China","institute":"Institute of Zoology","deparment":"National Research Center for Wildlife Born Diseases","abstract":"Human infection with avian influenza A H5N1 viruses can cause severe diseases with high mortality rate and continues to pose a significant threat to global public health. Rapid diagnosis is needed for identifying the types of influenza viruses for making timely treatment decisions. Here, we demonstrate absolute quantification of H5-subtype influenza viruses by digital loop-mediated isothermal amplification (dLAMP) on our recently developed cross-interface emulsification (XiE) method. Our results show that XiE-based dLAMP is highly specific and displays comparable sensitivity to real-time PCR (qPCR) and digital PCR (dPCR). Notably, dLAMP is more tolerant to inhibitory substances than PCR methods and demonstrated similar detection efficiency to qPCR for real H5N1 samples. Therefore, it can serve as a robust and precise alternative to qPCR or dPCR and is especially suitable for environmental and clinical samples with hard-to-remove contaminants. We believe that our dLAMP method offers great potential for rapid and accurate diagnosis of influenza and other infectious diseases.","lamp_id":[{"id":"LPB00288","pathogen":"Influenza A virus (H5N1)","target":"HA"}]},{"id":151,"pmid":26378283,"title":"Rapid Detection of Subtype H10N8 Influenza Virus by One-Step Reverse Transcription\u2013Loop-Mediated Isothermal Amplification Methods","year":2015,"journal":"Journal of Clinical Microbiology","authors":"Hongmei Bao, Xiaoxiao Feng, Yong Ma, Jianzhong Shi, Yuhui Zhao, Linlin Gu, Xiurong Wang, Hualan Chen","doi":"10.1128\/JCM.02165-15","country":"People's Republic of China","institute":"Harbin Veterinary Research Institute","deparment":"Animal Influenza Laboratory of the Ministry of Agriculture","abstract":"We developed hemagglutinin- and neuraminidase-specific one-step reverse transcription\u2013loop-mediated isothermal amplification assays for detecting the H10N8 virus. The detection limit of the assays was 10 copies of H10N8 virus, and the assays did not amplify nonspecific RNA. The assays can detect H10N8 virus from chicken samples with high sensitivity and specificity, and they can serve as an effective tool for detecting and monitoring H10N8 virus in live poultry markets.","lamp_id":[{"id":"LPB00289","pathogen":"Influenza A virus (H10N8)","target":"HA"},{"id":"LPB00290","pathogen":"Influenza A virus (H10N8)","target":"NA"}]},{"id":152,"pmid":23718218,"title":"Visual detection of human infection with influenza A (H7N9) virus by subtype-specific reverse transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye","year":2013,"journal":"Clinical Microbiology and Infection","authors":"K Nie, X Zhao, X Ding, X D Li, S M Zou, J F Guo, D Y Wang, R B Gao, X Y Li, W J Huang, Y L Shu, X J Ma","doi":"10.1111\/1469-0691.12263","country":"China","institute":"National Institute for Viral Disease Control and Prevention","deparment":"Key Laboratory for Medical Virology","abstract":"A rapid and sensitive H7 and N9 subtype-specific reverse transcription loop-mediated isothermal amplification assay was developed respectively for visual detection of human-infected influenza A (H7N9) virus. The reaction was performed in one step in a single tube at 63\u00b0C for 60 min with the addition of hydroxynaphthol blue dye before amplification. The detection limits of both subtype-specific assays were comparable to those of validated H7 and N9 real-time PCR assays respectively and no cross-detection was observed with influenza A pandemic H1N1, H3N2, H5N1, H9N2 or influenza B virus. The assays were evaluated further with H7N9 virus-infected clinical specimens.","lamp_id":[{"id":"LPB00291","pathogen":"Influenza A virus (H7N9)","target":"HA"},{"id":"LPB00292","pathogen":"Influenza A virus (H7N9)","target":"NA"}]},{"id":153,"pmid":33238709,"title":"Isothermal Amplification and Ambient Visualization in a Single Tube for the Detection of SARS-CoV-2 Using Loop-Mediated Amplification and CRISPR Technology","year":2020,"journal":"Analytical Chemistry","authors":"Bo Pang, Jingyang Xu, Yanming Liu, Hanyong Peng, Wei Feng, Yiren Cao, Jinjun Wu, Huyan Xiao, Kanti Pabbaraju, Graham Tipples, Michael A Joyce, Holly A Saffran, D Lorne Tyrrell, Hongquan Zhang, X Chris Le","doi":"10.1021\/acs.analchem.0c04047","country":"Canada","institute":"University of Alberta","deparment":"Department of Laboratory Medicine and Pathology","abstract":"We have developed a single-tube assay for SARS-CoV-2 in patient samples. This assay combined advantages of reverse transcription (RT) loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) enzyme Cas12a. Our assay is able to detect SARS-CoV-2 in a single tube within 40 min, requiring only a single temperature control (62 \u00b0C). The RT-LAMP reagents were added to the sample vial, while CRISPR Cas12a reagents were deposited onto the lid of the vial. After a half-hour RT-LAMP amplification, the tube was inverted and flicked to mix the detection reagents with the amplicon. The sequence-specific recognition of the amplicon by the CRISPR guide RNA and Cas12a enzyme improved specificity. Visible green fluorescence generated by the CRISPR Cas12a system was recorded using a smartphone camera. Analysis of 100 human respiratory swab samples for the N and\/or E gene of SARS-CoV-2 produced 100% clinical specificity and no false positive. Analysis of 50 samples that were detected positive using reverse transcription quantitative polymerase chain reaction (RT-qPCR) resulted in an overall clinical sensitivity of 94%. Importantly, this included 20 samples that required 30\u201339 threshold cycles of RT-qPCR to achieve a positive detection. Integration of the exponential amplification ability of RT-LAMP and the sequence-specific processing by the CRISPR-Cas system into a molecular assay resulted in improvements in both analytical sensitivity and specificity. The single-tube assay is beneficial for future point-of-care applications.","lamp_id":[{"id":"LPB00293","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00294","pathogen":"SARS-CoV-2","target":"E"}]},{"id":154,"pmid":15054079,"title":"Rapid Detection of the Severe Acute Respiratory Syndrome (SARS) Coronavirus by a Loop-Mediated Isothermal Amplification Assay","year":2004,"journal":"Clinical Chemistry","authors":"Leo L M Poon, Cynthia S W Leung, Masato Tashiro, Kwok Hung Chan, Bonnie W Y Wong, Kwok Yung Yuen, Yi Guan, Joseph S M Peiris","doi":"10.1373\/clinchem.2004.032011","country":"Hong Kong SAR","institute":"University of Hong Kong","deparment":"Department of Microbiology","abstract":"No abstract available.","lamp_id":[{"id":"LPB00295","pathogen":"SARS-CoV","target":"ORF1b"}]},{"id":155,"pmid":26050646,"title":"A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers","year":2015,"journal":"Scientific Reports","authors":"Yan Du, Randall A. Hughes, Sanchita Bhadra, Yu Sherry Jiang, Andrew D. Ellington, Bingling Li","doi":"10.1038\/srep11039","country":"USA","institute":"The University of Texas at Austin","deparment":"Center for Systems and Synthetic Biology","abstract":"Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20\u2013100 copies\/\u03bcl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device.","lamp_id":[{"id":"LPB00296","pathogen":"MERS-CoV","target":"ORF1a"}]},{"id":156,"pmid":33750853,"title":"Field-deployable, rapid diagnostic testing of saliva for SARS-CoV-2","year":2021,"journal":"Scientific Reports","authors":"Shan Wei, Hemant Suryawanshi, Alexandre Djandji, Esther Kohl, Stephanie Morgan, Eldad A. Hod, Susan Whittier, Kevin Roth, Raymond Yeh, Juan Carlos Alejaldre, Elaine Fleck, Stephen Ferrara, Daniel Hercz, David Andrews, Lilly Lee, Kristopher A. Hendershot, Joshua Goldstein, Yousin Suh, Mahesh Mansukhani, Zev Williams","doi":"10.1038\/s41598-021-84792-8","country":"USA","institute":"Columbia University Irving Medical Center","deparment":"","abstract":"To safely re-open economies and prevent future outbreaks, rapid, frequent, point-of-need, SARS-CoV-2 diagnostic testing is necessary. However, existing field-deployable COVID-19 testing methods require the use of uncomfortable swabs and trained providers in PPE, while saliva-based methods must be transported to high complexity laboratories for testing. Here, we report the development and clinical validation of High-Performance Loop-mediated isothermal Amplification (HP-LAMP), a rapid, saliva-based, SARS-CoV-2 test with a limit of detection of 1.4 copies of virus per \u00b5l of saliva and a sensitivity and specificity with clinical samples of\u2009>\u200996%, on par with traditional RT-PCR based methods using swabs, but can deliver results using only a single fluid transfer step and simple heat block. Testing of 120 patient samples in 40 pools comprised of 5 patient samples each with either all negative or a single positive patient sample was 100% accurate. Thus, HP-LAMP may enable rapid and accurate results in the field using saliva, without need of a high-complexity laboratory.","lamp_id":[{"id":"LPB00299","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":157,"pmid":33808752,"title":"Specific Detection of Influenza A and B Viruses by CRISPR-Cas12a-Based Assay","year":2021,"journal":"Biosensors","authors":"Bum Ju Park, Man Seong Park, Jae Myun Lee, Yoon Jae Song","doi":"10.3390\/bios11030088","country":"Korea","institute":"Gachon University","deparment":"Department of Life Science","abstract":"A rapid and accurate on-site diagnostic test for pathogens including influenza viruses is critical for preventing the spread of infectious diseases. Two types of influenza virus, A and B cause seasonal flu epidemics, whereas type A can cause influenza pandemics. To specifically detect influenza A (IAV) and B (IBV) viruses, we developed a clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system-based assay. By coupling reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), a CRISPR-Cas12a DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) detected IAV and IBV titers as low as 1 \u00d7 10^0 plaque forming units (PFUs) per reaction without exhibiting cross-reactivity. Only 75 to 85 min were required to detect IAV and IBV, depending on isothermal nucleic acid amplification methods, and results were verified using a lateral flow strip assay that does not require additional analytic equipment. Taken together, our findings establish RT-RPA and RT-LAMP-coupled DETECTR-based diagnostic tests for rapid, specific and high-sensitivity detection of IAV and IBV using fluorescence and lateral flow assays. The diagnostic test developed in this study can be used to distinguish IAV and IBV infections, a capability that is necessary for monitoring and preventing the spread of influenza epidemics and pandemics.","lamp_id":[{"id":"LPB00300","pathogen":"Influenza B virus","target":"HA"},{"id":"LPB00301","pathogen":"Influenza A virus (H1N1)","target":"M"}]},{"id":158,"pmid":34069710,"title":"Optimization and Clinical Evaluation of a Multi-Target Loop-Mediated Isothermal Amplification Assay for the Detection of SARS-CoV-2 in Nasopharyngeal Samples","year":2021,"journal":"Viruses","authors":"Foteini Roumani, Sarah Azinheiro, Hugo Sousa, Ana Sousa, Mafalda Tim\u00f3teo, Tatiana Varandas, Daniela Fonseca-Silva, In\u00eas Baldaque, Joana Carvalho, Marta Prado, Alejandro Garrido-Maestu","doi":"10.3390\/v13050940","country":"Portugal\/Spain","institute":"International Iberian Nanotechnology Laboratory\/University of Santiago de Compostela","deparment":"Food Quality and Safety Research Group\/Department of Analytical Chemistry, Nutrition and Food Science","abstract":"SARS-CoV-2 is the coronavirus responsible for COVID-19, which has spread worldwide, affecting more than 200 countries, infecting over 140 million people in one year. The gold standard to identify infected people is RT-qPCR, which is highly sensitive, but needs specialized equipment and trained personnel. The demand for these reagents has caused shortages in certain countries. Isothermal nucleic acid techniques, such as loop-mediated isothermal amplification (LAMP) have emerged as an alternative or as a complement to RT-qPCR. In this study, we developed and evaluated a multi-target RT-LAMP for the detection of SARS-CoV-2. The method was evaluated against an RT-qPCR in 152 clinical nasopharyngeal swab samples. The results obtained indicated that both assays presented a \u201cgood concordance\u201d (Cohen\u2019s k of 0.69), the RT-LAMP was highly specific (99%) but had lower sensitivity compared to the gold standard (63.3%). The calculated low sensitivity was associated with samples with very low viral load (RT-qPCR Cq values higher than 35) which may be associated with non-infectious individuals. If an internal Cq threshold below 35 was set, the sensitivity and Cohen\u2019s k increased to 90.9% and 0.92, respectively. The interpretation of the Cohen\u2019s k for this was \u201cvery good concordance\u201d. The RT-LAMP is an attractive approach for frequent individual testing in decentralized setups.","lamp_id":[{"id":"LPB00303","pathogen":"SARS-CoV-2","target":"ORF8"},{"id":"LPB00304","pathogen":"SARS-CoV-2","target":"ORF3a"},{"id":"LPB00305","pathogen":"SARS-CoV-2","target":"N"}]},{"id":159,"pmid":34075364,"title":"Intrinsic Signal Amplification by Type-III CRISPR-Cas Systems Provides a Sequence-Specific SARS-CoV-2 Diagnostic","year":2021,"journal":"Cell Reports Medicine","authors":"Andrew Santiago-Frangos, Laina N. Hall, Anna Nemudraia, Artem Nemudryi, Pushya Krishna, Tanner Wiegand, Royce A. Wilkinson, Deann T. Snyder, Jodi F. Hedges, Calvin Cicha, Helen H. Lee, Ava Graham, Mark A. Jutila, Matthew P. Taylor, Blake Wiedenheft","doi":"10.1016\/j.xcrm.2021.100319","country":"USA","institute":"Montana State University","deparment":"Department of Microbiology and Immunology","abstract":"There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 10^7 copies\/\u03bcL for a single guide RNA to 10^6 copies\/\u03bcL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies\/\u03bcL and is completed using patient samples in less than 30 min.","lamp_id":[{"id":"LPB00306","pathogen":"SARS-CoV-2","target":"N"}]},{"id":160,"pmid":34022500,"title":"Rapid and highly sensitive one-tube colorimetric RT-LAMP assay for visual detection of SARS-CoV-2 RNA","year":2021,"journal":"Biosensors and Bioelectronics","authors":"Yugan He, Tie Xie, Yigang Tong","doi":"10.1016\/j.bios.2021.113330","country":"People's Republic of China","institute":"Beijing University of Chemical Technology","deparment":"Beijing Advanced Innovation Center for Soft Matter Science and Engineering","abstract":"Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a highly contagious disease. To tame the continuously raging outbreak of COVID-19, developing a cheap, rapid and sensitive testing assay is absolutely imperative. Herein, we developed a one-tube colorimetric RT-LAMP assay for the visual detection of SARS-CoV-2 RNA. The assay integrates Si\u2013OH magnetic beads (MBs)-based fast RNA extraction and rapid isothermal amplification in a single tube, thus bypassing the RNA elution step and directly amplifying on-beads RNA molecules with the visualized results. This one-tube assay has a limit of detection (LOD) as low as 200 copies\/mL for sample input volumes of up to 600 \u03bcL, and can be performed in less than 1 h from sample collection to result readout. This assay demonstrated a 100% concordance with the gold standard test RT-qPCR test by using 29 clinical specimens and showed high specificity. This one-tube colorimetric RT-LAMP assay can serve as an alternative platform for a rapid and sensitive diagnostic test for COVID-19 and is particularly suitable for use at community clinics or township hospitals.","lamp_id":[{"id":"LPB00307","pathogen":"SARS-CoV-2","target":"RdRp"}]},{"id":161,"pmid":21729297,"title":"Visual detection of H3 subtype avian influenza viruses by reverse transcription loop-mediated isothermal amplification assay","year":2011,"journal":"Virology Journal","authors":"Yi Peng, Zhixun Xie, Jiabo Liu, Yaoshan Pang, Xianwen Deng, Zhiqin Xie, Liji Xie, Qing Fan, Jiaxun Feng,  Mazhar I Khan","doi":"10.1186\/1743-422X-8-337","country":"China","institute":"Guangxi Veterinary Research Institute","deparment":"Department of Biotechnology","abstract":"Background\nRecent epidemiological investigation of different HA subtypes of avian influenza viruses (AIVs) shows that the H3 subtype is the most predominant among low pathogenic AIVs (LPAIVs), and the seasonal variations in isolation of H3 subtype AIVs are consistent with that of human H3 subtype influenza viruses. Consequently, the development of a rapid, simple, sensitive detection method for H3 subtype AIVs is required. The loop-mediated isothermal amplification (LAMP) assay is a simple, rapid, sensitive and cost-effective nucleic acid amplification method that does not require any specialized equipment.\n\nResults\nA reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect the H3 subtype AIVs visually. Specific primer sets target the sequences of the hemagglutinin (HA) gene of H3 subtype AIVs were designed, and assay reaction conditions were optimized. The established assay was performed in a water bath for 50 minutes, and the amplification result was visualized directly as well as under ultraviolet (UV) light reflections. The detection limit of the RT-LAMP assay was 0.1pg total RNA of virus, which was one hundred-fold higher than that of RT-PCR. The results on specificity indicated that the assay had no cross-reactions with other subtype AIVs or avian respiratory pathogens. Furthermore, a total of 176 clinical samples collected from birds at the various live-bird markets (LBMs) were subjected to the H3-subtype-specific RT-LAMP (H3-RT-LAMP). Thirty-eight H3 subtype AIVs were identified from the 176 clinical samples that were consistent with that of virus isolation.\n\nConclusions\nThe newly developed H3-RT-LAMP assay is simple, sensitive, rapid and can identify H3 subtype AIVs visually. Consequently, it will be a very useful screening assay for the surveillance of H3 subtype AIVs in underequipped laboratories as well as in field conditions.","lamp_id":[{"id":"LPB00309","pathogen":"Influenza A virus (H3N2)","target":"HA"}]},{"id":162,"pmid":21155764,"title":"M-specific reverse transcription loop-mediated isothermal amplification for detection of pandemic (H1N1) 2009 virus","year":2010,"journal":"European Journal of Clinical Investigation","authors":"Min-Shiuh Lee, Hung-Chang Shih, Jang-Jih Lu, Mei-Chi Su, Ming-Chung Deng, Chia-Chen Wu, Fong-Yuan Lin, Kuan-Hsun Lin, Po-Yen Chen, Wei-Li Hsu","doi":"10.1111\/j.1365-2362.2010.02427.x","country":"Taiwan","institute":"Council of Agriculture","deparment":"Animal Health Research Institute","abstract":"Background: Since the initial outbreak in March 2009, the novel pandemic (H1N1) 2009 virus has affected individuals worldwide and caused over 18,138 deaths. There is an urgent need for the development of an easy, accurate and simple method for the diagnosis of this novel pandemic virus.\n\nDesign: Reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) with primers targeting the M segment was established for the rapid differential diagnosis of pandemic (H1N1) 2009 virus. The performance of this assay was characterized using 111 clinic nasopharyngeal swabs, and the diagnosis accuracy was compared with real-time reverse transcription PCR (RRT-PCR) and virus isolation, the latter being the reference standard.\n\nResults: This method successfully detected pandemic (H1N1) 2009 virus with a detection limit of approximately 20 copies of the target RNA per reaction, which is a comparably sensitivity to the RRT-PCR assay. Furthermore, this assay was able to discriminate pandemic (H1N1) 2009 virus from seasonal influenza viruses, such as H1N1 and H3N2, and other respiratory viruses (parainfluenza type 2 and 3, adenovirus, echovirus 7, and coxsackievirus A10). Based on validation by virus isolation, the specificity and sensitivity of this M-specific RT-LAMP assay were 100% and 98\u00b725%, respectively. Moreover, the RT-LAMP amplification of most positive samples (46 out of 56) was achieved in < 20 min.\n\nConclusions: This is an accurate and fast analysis system suitable for general diagnostic laboratories with only limited equipment, e.g. first-line health care centre. This assay will help clinicians and public health officials to react effectively during an outbreak.","lamp_id":[{"id":"LPB00310","pathogen":"Influenza A virus (H1N1)","target":"M"}]},{"id":163,"pmid":24269205,"title":"Identification of human metapneumovirus genotypes A and B from clinical specimens by reverse transcription loop-mediated isothermal amplification","year":2014,"journal":"Journal of Virological Methods","authors":"Qinwei Song, Runan Zhu, Yu Sun, Linqing Zhao, Fang Wang, Jie Deng, Yuan Qian","doi":"10.1016\/j.jviromet.2013.10.037","country":"China","institute":"Capital Institute of Pediatrics","deparment":"Laboratory of Virology","abstract":"Human metapneumovirus (hMPV) has been recognized as an important pathogen for acute respiratory infections in children worldwide and classified into genotypes A and B. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay is a rapid diagnostic method for detecting nucleic acids with a single step under isothermal conditions in less than 1 h. RT-LAMP targeting the M gene of hMPV was developed for detecting and identifying hMPV genotypes A and B. The detection limit of the genotype-specific hMPV RT-LAMP assay was 10 times greater than that of conventional reverse transcription polymerase chain reaction (RT-PCR). No cross-reactivity was found with respiratory syncytial virus, parainfluenza virus 1\u20133, adenovirus, human bocavirus, human rhinovirus, influenza virus A and B, human coronaviruses and enteroviruses. One hundred and fifteen clinical specimens were detected for hMPV genotypes A and B with RT-LAMP, RT-PCR and real-time SYBR PCR. Kappa coefficients showed that there was a good agreement among these three methods. Compared with RT-PCR and real-time SYBR PCR, the genotype-specific RT-LAMP showed better specificity, sensitivity and is more convenient to perform with reduced turn-around time.","lamp_id":[{"id":"LPB00311","pathogen":"HMPV","target":"M"},{"id":"LPB00312","pathogen":"HMPV","target":"M"}]},{"id":164,"pmid":27376196,"title":"An immunoassay-based reverse-transcription loop-mediated isothermal amplification assay for the rapid detection of avian influenza H5N1 virus viremia","year":2016,"journal":"Biosensors and Bioelectronics","authors":"Yi Tang, Xu Yu, Hao Chen, Youxiang Diao","doi":"10.1016\/j.bios.2016.06.063","country":"China","institute":"Shandong Agricultural University","deparment":"College of Animal Science and Veterinary Medicine","abstract":"Avian influenza virus (AIV) subtype H5N1 attracts particular consideration because it is a continuous threat to animals and public health systems. The viremia caused by AIV H5N1 infection may increase the risk of blood-borne transmission between humans. Therefore, there is a need to rapidly evaluate and implement screening measures for AIV H5N1 viremia that allows for rapid response to this potentially pandemic threat. The present report describes an immunoassay-based reverse-transcription loop-mediated isothermal amplification (immuno-RT-LAMP) assay for the rapid detection of AIV H5N1 in whole blood samples. Using PCR tubes coated with an H5 subtype monoclonal antibody, AIV H5N1 virions were specifically captured from blood samples. After a thermal lysis step, the released viral N1 gene was exponentially amplified using RT-LAMP on either a real-time PCR instrument for quantitative analysis, or in a water bath system for endpoint analysis. The detection limit of the newly developed immuno-RT-LAMP assay was as low as 1.62\u00d710^1 50% embryo infectious dose\/mL of virus in both regular samples and simulated viremia samples. There were no cross-reactions with non-H5N1 influenza viruses or other avian viruses. The reproducibility of the assay was confirmed using intra- and inter-assay tests with variability ranging from 1.05% to 3.37%. Our results indicate that immuno-RT-LAMP is a novel, effective point-of-care virus identification solution for the rapid diagnosis and monitoring of AIV H5N1 in blood samples.","lamp_id":[{"id":"LPB00313","pathogen":"Influenza A virus (H5N1)","target":"NA"}]},{"id":165,"pmid":24648558,"title":"Improved detection limit in rapid detection of human enterovirus 71 and coxsackievirus A16 by a novel reverse transcription-isothermal multiple-self-matching-initiated amplification assay","year":2014,"journal":"Journal of Clinical Microbiology","authors":"Xiong Ding, Kai Nie, Lei Shi, Yong Zhang, Li Guan, Dan Zhang, Shunxiang Qi, Xuejun Ma","doi":"10.1128\/JCM.03298-13","country":"China","institute":"South China University of Technology\/National Institute for Viral Disease Control and Prevention","deparment":"School of Light Industry and Food Sciences\/Key Laboratory for Medical Virology","abstract":"Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-foot-and-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription-isothermal multiple-self-matching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R(2) values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R(2) values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies\/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies\/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71 and CVA16 viruses.","lamp_id":[{"id":"LPB00314","pathogen":"EV-A71","target":"VP1"},{"id":"LPB00324","pathogen":"CVA16","target":"VP1"}]},{"id":166,"pmid":33428641,"title":"Performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification","year":2021,"journal":"PLoS One","authors":"Pierre Garneret, Etienne Coz, Elian Martin, Jean-Claude Manuguerra, Elodie Brient-Litzler, Vincent Enouf, Daniel Felipe Gonz\u00e1lez Obando, Jean-Christophe Olivo-Marin, Fabrice Monti, Sylvie van der Werf, Jessica Vanhomwegen, Patrick Tabeling","doi":"10.1371\/journal.pone.0243712","country":"France","institute":"ESPCI PSL","deparment":"IPGG","abstract":"To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT-PCR (Reverse transcription-Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings [1]. In the present work, by harnessing progress made in the past two decades in isothermal amplification and paper microfluidics, we created a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT-LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or fluorescent probes. Depending on the viral load in the tested samples, the detection takes between twenty minutes and one hour. Using a set of 16 pools of naso-pharyngal swab eluates, we estimated a limit of detection comparable to real-time RT-PCR (i.e. 1 genome copies per microliter of clinical sample) and no cross-reaction with eight major respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called \"COVIDISC\" to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment will expedite the widespread dissemination of this device. What is proposed here is a new efficient tool to help managing the pandemics.","lamp_id":[{"id":"LPB00315","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"}]},{"id":167,"pmid":33545551,"title":"Sequence-specific and multiplex detection of COVID-19 virus (SARS-CoV-2) using proofreading enzyme-mediated probe cleavage coupled with isothermal amplification","year":2021,"journal":"Biosensors and Bioelectronics","authors":"Sheng Ding, Gangyi Chen, Yinghua Wei, Juan Dong, Feng Du, Xin Cui, Xin Huang, Zhuo Tang","doi":"10.1016\/j.bios.2021.113041","country":"People's Republic of China","institute":"Chengdu Institute of Biology\/University of Chinese Academy of Sciences","deparment":"Natural Products Research Center","abstract":"The outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been challenging human health worldwide. Loop-mediated isothermal amplification (LAMP) has been promptly applied to the detection of SARS-CoV-2 owing to its high amplification efficacy and less requirement of the thermal cycler. However, the vast majority of these LAMP-based assays depend on the non-specific detection of LAMP products, which can not discern the undesirable amplificons, likely to yield unreliable results. Herein, a sequence-specific LAMP assay was reported to detect SARS-CoV-2 using proofreading enzyme-mediated probe cleavage (named Proofman), which could realize real-time and visual detection without uncapping. This assay, introducing a proofreading enzyme and the fluorogenic probe to reverse-transcription LAMP (RT-Proofman-LAMP), can specifically detect the SARS-CoV-2 RNA with a detection limit of 100 copies. In addition to the real-time analysis, the assay is capable of endpoint visualization under a transilluminator within 50 min, providing a convenient reporting manner under the setting of point-of-care testing (POCT). In combination with different fluorophores, the one-pot multiplex assay was successfully achieved to detect multiple targets of SARS-CoV-2 and inner control simultaneously. In summary, the development of RT-Proofman-LAMP offers a versatile and highly-specific method for fast field screening and laboratory testing of SARS-CoV-2, making it a promising platform in COVID-19 diagnosis.","lamp_id":[{"id":"LPB00316","pathogen":"SARS-CoV-2","target":"N"}]},{"id":168,"pmid":36445095,"title":"Development and Validation of a Novel COVID-19 nsp8 One-Tube RT-LAMP-CRISPR Assay for SARS-CoV-2 Diagnosis","year":2022,"journal":"Microbiology Spectrum","authors":"Cyril Chik-Yan Yip, Siddharth Sridhar, Wan-Mui Chan, Jonathan Daniel Ip, Allen Wing-Ho Chu, Kit-Hang Leung, Vincent Chi-Chung Cheng, Kwok-Yung Yuen, Kelvin Kai-Wang To","doi":"10.1128\/spectrum.01962-22","country":"","institute":"","deparment":"","abstract":"Accurate and simple diagnostic tests for coronavirus disease 2019 (COVID-19) are essential components of the pandemic response. In this study, we evaluated a one-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay coupled with clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-mediated endpoint detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in clinical samples. RT-LAMP-CRISPR is fast and affordable, does not require bulky thermocyclers, and minimizes carryover contamination risk. Results can be read either visually or with a fluorometer. RT-LAMP-CRISPR assays using primers targeting a highly expressed nsp8 gene and previously described nucleocapsid (N) gene primers were designed. The analytical characteristics and diagnostic performance of RT-LAMP-CRISPR assays were compared to those of a commercial real-time RT-PCR E gene assay. The limits of detection (LODs) of the nsp8 and N RT-LAMP-CRISPR assays were 750 and 2,000 copies\/mL, which were higher than that of the commercial real-time RT-PCR assay (31.3 copies\/mL). Despite the higher LOD, RT-LAMP-CRISPR assays showed diagnostic sensitivity and specificity of 98.6% and 100%, respectively, equivalent to those of the real-time RT-PCR assay (P = 0.5). The median fluorescence reading from the nsp8 assay (378.3 raw fluorescence unit [RFU] [range, 215.6 to 592.6]) was significantly higher than that of the N gene assay (342.0 RFU [range, 143.0 to 576.6]) (P < 0.0001). In conclusion, we demonstrate that RT-LAMP-CRISPR assays using primers rationally designed from highly expressed gene targets are highly sensitive, specific, and easy to perform. Such assays are a valuable asset in resource-limited settings.","lamp_id":[{"id":"LPB00317","pathogen":"SARS-CoV-2","target":"NSP8"},{"id":"LPB00676","pathogen":"SARS-CoV-2","target":"N"}]},{"id":169,"pmid":32999292,"title":"Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection","year":2020,"journal":"Nature Communications","authors":"Long T. Nguyen, Brianna M. Smith, Piyush K. Jain","doi":"10.1038\/s41467-020-18615-1","country":"USA","institute":"University of Florida","deparment":"Department of Chemical Engineering","abstract":"The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the femtomolar range without any target pre-amplification step. By extending the 3'- or 5'-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discover a self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40-60 min.","lamp_id":[{"id":"LPB00318","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00319","pathogen":"SARS-CoV-2","target":"N"}]},{"id":170,"pmid":33674580,"title":"Point-of-care bulk testing for SARS-CoV-2 by combining hybridization capture with improved colorimetric LAMP","year":2021,"journal":"Nature Communications","authors":"Lukas Bokelmann, Olaf Nickel, Tomislav Maricic, Svante P\u00e4\u00e4bo, Matthias Meyer, Stephan Borte, Stephan Riesenberg","doi":"10.1038\/s41467-021-21627-0","country":"Germany","institute":"Max Planck Institute for Evolutionary Anthropology","deparment":"","abstract":"Efforts to contain the spread of SARS-CoV-2 have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can be applied to large numbers of people. However, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation and sophisticated laboratory equipment to achieve throughputs that allow whole communities to be tested on a regular basis. Here we present Cap-iLAMP (capture and improved loop-mediated isothermal amplification) which combines a hybridization capture-based RNA extraction of gargle lavage samples with an improved colorimetric RT-LAMP assay and smartphone-based color scoring. Cap-iLAMP is compatible with point-of-care testing and enables the detection of SARS-CoV-2 positive samples in less than one hour. In contrast to direct addition of the sample to improved LAMP (iLAMP), Cap-iLAMP prevents false positives and allows single positive samples to be detected in pools of 25 negative samples, reducing the reagent cost per test to ~1 Euro per individual.","lamp_id":[{"id":"LPB00320","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"},{"id":"LPB00321","pathogen":"SARS-CoV-2","target":"N"}]},{"id":171,"pmid":null,"title":"Clinical Assessment and Validation of a Rapid and Sensitive SARS-CoV-2 Test Using Reverse Transcription Loop-Mediated Isothermal Amplification Without the Need for RNA Extraction","year":2020,"journal":"Open Forum Infectious Diseases","authors":"Melis N Anahtar, Graham E G McGrath, Brian A Rabe, Nathan A Tanner, Benjamin A White, Jochen K M Lennerz, John A Branda, Constance L Cepko, Eric S Rosenberg","doi":"10.1093\/ofid\/ofaa631","country":"USA","institute":"Massachusetts General Hospital and Harvard Medical School\/Massachusetts General Hospital\/Blavatnik Institute\/New England Biolabs\/Howard Hughes Medical Institute","deparment":"Department of Pathology\/Department of Medicine\/Department of Genetics\/Department of Ophthalmology\/Department of Emergency Medicine","abstract":"Amid the enduring pandemic, there is an urgent need for expanded access to rapid, sensitive, and inexpensive coronavirus disease 2019 (COVID-19) testing worldwide without specialized equipment. We developed a simple test that uses colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect severe acute resrpiratory syndrome coronavirus 2 (SARS-CoV-2) in 40 minutes from sample collection to result.\n\nMethods\nWe tested 135 nasopharyngeal specimens from patients evaluated for COVID-19 infection at Massachusetts General Hospital. Specimens were either added directly to RT-LAMP reactions, inactivated by a combined chemical and heat treatment step, or inactivated then purified with a silica particle\u2013based concentration method. Amplification was performed with 2 SARS-CoV-2-specific primer sets and an internal specimen control; the resulting color change was visually interpreted.\n\nResults\nDirect RT-LAMP testing of unprocessed specimens could only reliably detect samples with abundant SARS-CoV-2 (>3 000 000 copies\/mL), with sensitivities of 50% (95% CI, 28%\u201372%) and 59% (95% CI, 43%\u201373%) in samples collected in universal transport medium and saline, respectively, compared with quantitative polymerase chain reaction (qPCR). Adding an upfront RNase inactivation step markedly improved the limit of detection to at least 25 000 copies\/mL, with 87.5% (95% CI, 72%\u201395%) sensitivity and 100% specificity (95% CI, 87%\u2013100%). Using both inactivation and purification increased the assay sensitivity by 10-fold, achieving a limit of detection comparable to commercial real-time PCR-based diagnostics.\n\nConclusions\nBy incorporating a fast and inexpensive sample preparation step, RT-LAMP accurately detects SARS-CoV-2 with limited equipment for about US$6 per sample, making this a potentially ideal assay to increase testing capacity, especially in resource-limited settings.","lamp_id":[{"id":"LPB00322","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00323","pathogen":"SARS-CoV-2","target":"N"}]},{"id":172,"pmid":33922716,"title":"Accessible LAMP-Enabled Rapid Test (ALERT) for Detecting SARS-CoV-2","year":2021,"journal":"Viruses","authors":"Ali Bekta\u015f, Michael F Covington, Guy Aidelberg, Anibal Arce, Tamara Matute, Isaac N\u00fa\u00f1ez, Julia Walsh, David Boutboul, Constance Delaugerre, Ariel B Lindner, Fern\u00e1n Federici, Anitha D Jayaprakash","doi":"10.3390\/v13050742","country":"USA","institute":"Oakland Genomics Center","deparment":"","abstract":"The coronavirus disease 2019 (COVID-19) pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. Polymerase chain reaction (PCR)-based diagnostic tests are expensive, reliant on centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests that bind and detect the surface proteins of a virus are rapid and scalable but suffer from high false negative rates. To address this problem, an inexpensive, simple, and robust 60-minute do-it-yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles\/\u03bcL) and specificity (>97% true negative rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed. ALERT (Accessible LAMP-Enabled Rapid Test) incorporates the following features: (1) increased shelf-life and ambient temperature storage, compared to liquid reaction mixes, by using wax layers to isolate enzymes from other reagents; (2) improved specificity compared to other LAMP end-point reporting methods, by using sequence-specific QUASR (quenching of unincorporated amplification signal reporters); (3) increased sensitivity, compared to methods without purification through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal; (4) quality control with a nasopharyngeal-specific mRNA target; and (5) co-detection of other respiratory viruses, such as influenza B, by multiplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for labs and hospitals. With minimal effort, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms, or nucleic acid-based markers.","lamp_id":[{"id":"LPB00325","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00326","pathogen":"SARS-CoV-2","target":"N"}]},{"id":173,"pmid":34011690,"title":"High-Surety Isothermal Amplification and Detection of SARS-CoV-2","year":2021,"journal":"mSphere","authors":"Sanchita Bhadra, Timothy E Riedel, Simren Lakhotia, Nicholas D Tran, Andrew D Ellington","doi":"10.1128\/mSphere.00911-20","country":"USA","institute":"The University of Texas at Austin","deparment":"Department of Molecular Biosciences\/Center for Systems and Synthetic Biology","abstract":"Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva.","lamp_id":[{"id":"LPB00327","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00328","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00329","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"}]},{"id":174,"pmid":null,"title":"Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method","year":2020,"journal":"medRxiv","authors":"Weihua Yang, Xiaofei Dang, Qingxi Wang, Mingjie Xu, Qianqian Zhao, Yunying Zhou, Huailong Zhao, Li Wang, Yihui Xu, Jun Wang, Shuyi Han, Min Wang, Fenyan Pei, Yunshan Wan","doi":"10.1101\/2020.03.02.20030130","country":"China","institute":"Jinan central hospital affiliated to shandong first medical university","deparment":"Department of clinical laboratory","abstract":"Corona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real-time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 \u00b0C isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.","lamp_id":[{"id":"LPB00330","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00331","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00332","pathogen":"SARS-CoV-2","target":"E"}]},{"id":175,"pmid":null,"title":"A Simple Colorimetric Molecular Detection of Novel Coronavirus (COVID-19), an Essential Diagnostic Tool for Pandemic Screening","year":2020,"journal":"medRxiv","authors":"Pazhanimuthu Annamalai, Madhu Kanta, Pazhanivel Ramu, Baskar Ravi, Kokilavani Veerapandian, Rengarajan Srinivasan","doi":"10.1101\/2020.04.10.20060293","country":"India","institute":"Aura Biotechnologies Private Limited","deparment":"","abstract":"The recent outbreak of the newly emerged novel coronavirus (SARS-CoV-2) presents a big challenge for public health laboratories as virus isolates are not available while there is an increasing evidence that the epidemic is more widespread than initially thought, as well as spreading internationally across borders through travellers does already happen warranting a methodology for the rapid detection of the infection to control SARS-CoV-2. Aim: We intended to develop and deploy a robust and rapid diagnostic methodology using LAMP assay for use in point of care settings to detect SARS-COV-2 infection. Methodology: In the present study, we have developed a validated rapid diagnostic procedure to detect SARS-CoV-2 using LAMP assay, its design relying on isothermal amplification of the nucleic acids of the SARS-CoV-2. Results: The LAMP assay developed detects SARS-CoV-2 infection rapidly with high sensitivity and reliability. The data generated by LAMP assay were comparable and at par with the data generated by real-time PCR method. Conclusion: The present study demonstrates that the LAMP assay developed was a rapid, reliable, sensitive and cost effective method to detect SARS-CoV-2 infection in a point of care as well as in laboratory settings.","lamp_id":[{"id":"LPB00333","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00334","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":176,"pmid":35532013,"title":"A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva","year":2022,"journal":"eLife","authors":"Robert Warneford-Thomson, Parisha P Shah, Patrick Lundgren, Jonathan Lerner, Jason Morgan, Antonio Davila, Benjamin S Abella, Kenneth Zaret, Jonathan Schug, Rajan Jain, Christoph A Thaiss, Roberto Bonasio","doi":"10.7554\/eLife.69949","country":"USA","institute":"University of Pennsylvania Perelman School of Medicin","deparment":"Graduate Group in Biochemistry and Biophysics\/Epigenetics Institute\/Department of Cell and Developmental Biology","abstract":"The COVID-19 pandemic has created an urgent need for rapid, effective, and low-cost SARS-CoV-2 diagnostic testing. Here, we describe COV-ID, an approach that combines RT-LAMP with deep sequencing to detect SARS-CoV-2 in unprocessed human saliva with a low limit of detection (5-10 virions). Based on a multi-dimensional barcoding strategy, COV-ID can be used to test thousands of samples overnight in a single sequencing run with limited labor and laboratory equipment. The sequencing-based readout allows COV-ID to detect multiple amplicons simultaneously, including key controls such as host transcripts and artificial spike-ins, as well as multiple pathogens. Here, we demonstrate this flexibility by simultaneous detection of 4 amplicons in contrived saliva samples: SARS-CoV-2, influenza A, human STATHERIN, and an artificial SARS calibration standard. The approach was validated on clinical saliva samples, where it showed excellent agreement with RT-qPCR. COV-ID can also be performed directly on saliva absorbed on filter paper, simplifying collection logistics and sample handling.","lamp_id":[{"id":"LPB00335","pathogen":"SARS-CoV-2","target":"N"}]},{"id":177,"pmid":35027876,"title":"Saliva \u201cTreat-and-Heat\u201d Triplex Reverse Transcription Loop-Mediated Isothermal Amplification Assay for SARS-CoV-2","year":2021,"journal":"Journal of Biomolecular Techniques","authors":"Marie J. Y. Reolo, Carlos S. R. Eleazar, Joseph P. Sonio, Ryonne T. Solon, Janika L. B. Villamor, Alexandra K. Loedin, and Keith J. M. Moore","doi":"10.7171\/jbt.21-3203-014","country":"","institute":"","deparment":"","abstract":"The demand for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) molecular diagnostics that are faster, cheaper, and simpler to run than nasopharyngeal-based reverse transcription quantitative PCR (RT-qPCR) tests remains unmet in many parts of the world. In the Philippines, geographical and economic access to quality diagnostic testing remains out of reach for many communities. We describe the preclinical development of a fluorescence-based reverse transcription loop-mediated isothermal amplification test that uses drooled saliva as the biospecimen. Six treat-and-heat (\"direct\") procedures that inactivate the virus and release the target RNA were compared. Using duplexed As1e and E1 primers, protocols derived from Ben-Assa et al. (2020) using proteinase K or from Rabe and Cepko (2020) using TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)\/EDTA provided reliable RNA amplification. The TCEP\/EDTA-based method in particular showed improvement in robustness in duplex vs. singleplex format. Inclusion of human \u03b2-actin primers provided a triplex test with an internal amplification control that could be distinguished from SARS-CoV-2 amplicons based on melt curve analysis. After including the dUTP\/uracil-DNA glycosylase system and implementing laboratory procedures to avoid cross-contamination, false positive amplification was acceptably rare. The duplex or triplex tests are predicted to reliably detect patient salivary viral loads >100 copies\/\u03bcL and to yield equivocal results between 10 and 100 copies\/\u03bcL. These viral loads, corresponding to RT-qPCR C t \u223c29-32, are expected to identify the majority of infected and, particularly, of infectious patients. If clinically validated, the test would provide additional testing capacity requiring only a fraction of the time, cost, and infrastructure of the current nasopharyngeal swab-based RT-qPCR test, thereby improving access to testing for more Filipinos.","lamp_id":[{"id":"LPB00336","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00840","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":178,"pmid":34095506,"title":"SARS-CoV-2 detection by a clinical diagnostic RT-LAMP assay","year":2021,"journal":"Wellcome Open Research","authors":"Michael D Buck, Enzo Z Poirier, Ana Cardoso, Bruno Frederico, Johnathan Canton, Sam Barrell, Rupert Beale, Richard Byrne, Simon Caidan, Margaret Crawford, Laura Cubitt, Sonia Gandhi, Robert Goldstone, Paul R Grant, Kiran Gulati, Steve Hindmarsh, Michael Howell, Michael Hubank, Rachael Instrell, Ming Jiang, George Kassiotis, Wei-Ting Lu, James I MacRae, Iana Martini, Davin Miller, David Moore, Eleni Nastouli, Jerome Nicod, Luke Nightingale, Jessica Olsen, Amin Oomatia, Nicola O'Reilly, Anett Rideg, Ok-Ryul Song, Amy Strange, Charles Swanton, Samra Turajlic, Mary Wu, Caetano Reis e Sousa, Crick COVID-19 Consortium","doi":"10.12688\/wellcomeopenres.16517.2","country":"United Kingdom","institute":"The Francis Crick Institute","deparment":"","abstract":"The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARS-CoV-2 detection by RT-LAMP that is robust, reliable, repeatable, specific, and inexpensive.","lamp_id":[{"id":"LPB00337","pathogen":"SARS-CoV-2","target":"N"}]},{"id":179,"pmid":null,"title":"Handyfuge-LAMP: low-cost and electricity-free centrifugation for isothermal SARS-CoV-2 detection in saliva","year":2020,"journal":"medRxiv","authors":"Ethan Li, Adam Larson, Anesta Kothari, Manu Prakash","doi":"10.1101\/2020.06.30.20143255","country":"USA","institute":"Stanford University","deparment":"Department of Bioengineering","abstract":"Point of care diagnostics for COVID-19 detection are vital to assess infection quickly and at the source so appropriate measures can be taken. The loop-mediated isothermal amplification (LAMP) assay has proven to be a reliable and simple protocol that can detect small amounts of viral RNA in patient samples (<10 genomes per \u03bcL) (Nagamine, Hase, and Notomi 2002). Recently, Rabe and Cepko at Harvard published a sensitive and simple protocol for COVID-19 RNA detection in saliva using an optimized LAMP assay (Rabe and Cepko, 2020).\n\nThis LAMP protocol has the benefits of being simple, requiring no specialized equipment; rapid, requiring less than an hour from sample collection to readout; and cheap, costing around $1 per reaction using commercial reagents. The pH based colorimetric readout also leaves little ambiguity and is intuitive. However, a shortfall in many nucleic acid-based methods for detection in saliva samples has been the variability in output due to the presence of inhibitory substances in saliva. Centrifugation to separate the reaction inhibitors from inactivated sample was shown to be an effective way to ensure reliable LAMP amplification. However, a centrifuge capable of safely achieving the necessary speeds of 2000 RPM for several minutes often costs hundreds of dollars and requires a power supply.\n\nWe present here an open hardware solution- Handyfuge - that can be assembled with readily available components for the cost of <5 dollars a unit and could be used together with the LAMP assay for point of care detection of COVID-19 RNA from saliva. The device is then validated using the LAMP protocol from Rabe and Cepko. With the use of insulated coolers for reagent supply chain and delivery, the assay presented can be completed without the need for electricity or any laboratory scale infrastructure.","lamp_id":[{"id":"LPB00338","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00339","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":180,"pmid":35027872,"title":"Rapid, Affordable, and Scalable SARS-CoV-2 Detection From Saliva","year":2021,"journal":"Journal of Biomolecular Techniques","authors":"Andrew Hayden, Marcy Kuentzel, Sridar V. Chittur","doi":"10.7171\/jbt.21-3203-010","country":"USA","institute":"University at Albany","deparment":"Center for Functional Genomics","abstract":"Here we present an inexpensive, rapid, and robust reverse-transcription loop-mediated isothermal amplification (RT-LAMP)-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection method that is easily scalable, enabling point-of-care facilities and clinical labs to determine results from patients' saliva directly in 30 minutes for less than $2 per reaction. The method uses a novel combination of widely available reagents that can be prepared in bulk, plated, and frozen and remain stable until samples are received. This innovation dramatically reduces preparation time, enabling high-throughput automation and testing with time to results (including setup) in less than 1 hour for 96 patient samples simultaneously when using a 384-well format. By using a dual reporter (phenol red pH indicator for end-point detection and SYTO-9 fluorescent dye for real time), the assay also provides internal validation of results and redundancy in the event of an instrument malfunction.","lamp_id":[{"id":"LPB00340","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00341","pathogen":"SARS-CoV-2","target":"E"}]},{"id":181,"pmid":35273232,"title":"Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing","year":2022,"journal":"Scientific Reports","authors":"Kyle H Cole, Alexis Bouin, Caila Ruiz, Bert L Semler, Matthew A Inlay, Andrej Lupt\u00e1k","doi":"10.1038\/s41598-022-07871-4","country":"USA","institute":"University of California","deparment":"Department of Molecular Biology and Biochemistry","abstract":"The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 \u00b5L microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.","lamp_id":[{"id":"LPB00342","pathogen":"SARS-CoV-2","target":"N"}]},{"id":182,"pmid":35027867,"title":"Molecular Beacons Allow Specific RT-LAMP Detection of B.1.1.7 Variant SARS-CoV-2","year":2021,"journal":"Journal of Biomolecular Techniques","authors":"Scott Sherrill-Mix, Gregory D. Van Duyne, Frederic D. Bushman","doi":"10.7171\/jbt.21-3203-004","country":"USA","institute":"University of Pennsylvania","deparment":"Department of Microbiology\/Department of Medicine","abstract":"Over the course of the coronavirus disease 2019 (COVID-19) pandemic, several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic variants of concern have appeared and spread throughout the world. Detection and identification of these variants are important to understanding and controlling their rapid spread. Current detection methods for a particularly concerning variant, B.1.1.7, require expensive quantitative PCR machines and depend on the absence of a signal rather than a positive indicator of variant presence. Here we report an assay using a pair of molecular beacons combined with reverse transcription loop mediated amplification to allow isothermal amplification from saliva to specifically detect B.1.1.7 and other variants that contain a characteristic deletion in the gene encoding the viral spike protein. This assay is specific and affordable and allows multiplexing with other SARS-CoV-2 loop-mediated amplification primer sets.","lamp_id":[{"id":"LPB00343","pathogen":"SARS-CoV-2","target":"S"}]},{"id":183,"pmid":31005153,"title":"A low-cost microfluidic platform for rapid and instrument-free detection of whooping cough","year":2019,"journal":"Analytica Chimica Acta","authors":"Maowei Dou, Juan Sanchez, Hamed Tavakoli, Jeffrey E Gonzalez, Jianjun Sun, Jennifer Dien Bard, XiuJun Li","doi":"10.1016\/j.aca.2019.03.001","country":"","institute":"","deparment":"","abstract":"Whooping cough also called Pertussis is a highly contagious respiratory infection that affects all age populations. Given recent pertussis outbreaks, there is an urgent need for a point-of-care (POC) device for rapid diagnosis of pertussis. Herein, we report a low-cost microfluidic POC device integrated with loop-mediated isothermal amplification (LAMP) technique for the rapid and accurate diagnosis of pertussis. The 3D-printed bioanalyzer housed not only the biochip but also an in-house-developed portable and fully battery-powered heater for rapid POC detection of pertussis, without the need of external electricity. The fluorescence-based results could be rapidly visualized in about one hour by the naked eye without the need for any additional instrumentation. In addition, a simple centrifuge-free sample preparation process was optimized for the efficient lysis of pertussis samples and successfully used for direct detection of bacteria in nasopharyngeal samples. High sensitivity, with a limit of detection (LOD) of 5 DNA copies per LAMP zone, and high specificity were demonstrated. We envision that the microfluidic POC device can be used in various venues such as medical clinics, schools, and other low-resource settings for the fast detection of pertussis.","lamp_id":[{"id":"LPB00344","pathogen":"Bordetella pertussis Tohama I","target":"PT promoter region"}]},{"id":184,"pmid":35027873,"title":"Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared with endpoint assessment","year":2021,"journal":"Journal of Biomolecular Techniques","authors":"Lena M Diaz, Brandon E Johnson, Daniel M Jenkins","doi":"10.7171\/jbt.21-3203-011","country":"USA","institute":"University of Hawaii at Manoa","deparment":"Department of Molecular Biosciences and Bioengineering","abstract":"Controlling the course of the Coronavirus Disease 2019 (COVID-19) pandemic will require widespread deployment of consistent and accurate diagnostic testing of the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Ideally, tests should detect a minimum viral load, be minimally invasive, and provide a rapid and simple readout. Current Food and Drug Administration (FDA)-approved RT-qPCR-based standard diagnostic approaches require invasive nasopharyngeal swabs and involve laboratory-based analyses that can delay results. Recently, a loop-mediated isothermal nucleic acid amplification (LAMP) test that utilizes colorimetric readout received FDA approval. This approach utilizes a pH indicator dye to detect drop in pH from nucleotide hydrolysis during nucleic acid amplification. This method has only been approved for use with RNA extracted from clinical specimens collected via nasopharyngeal swabs. In this study, we developed a quantitative LAMP-based strategy to detect SARS-CoV-2 RNA in saliva. Our detection system distinguished positive from negative sample types using a handheld instrument that monitors optical changes throughout the LAMP reaction. We used this system in a streamlined LAMP testing protocol that could be completed in less than 2 h to directly detect inactivated SARS-CoV-2 in minimally processed saliva that bypassed RNA extraction, with a limit of detection (LOD) of 50 genomes\/reaction. The quantitative method correctly detected virus in 100% of contrived clinical samples spiked with inactivated SARS-CoV-2 at either 1\u00d7 (50 genomes\/reaction) or 2\u00d7 (100 genomes\/reaction) of the LOD. Importantly, the quantitative method was based on dynamic optical changes during the reaction and was able to correctly classify samples that were misclassified by endpoint observation of color.","lamp_id":[{"id":"LPB00345","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00346","pathogen":"SARS-CoV-2","target":"E"}]},{"id":185,"pmid":24456841,"title":"Rapid and simple detection of methicillin-resistance staphylococcus aureus by orfX loop-mediated isothermal amplification assay","year":2014,"journal":"BMC Biotechnology","authors":"Jianyu Su, Xiaochen Liu, Hemiao Cui, Yanyan Li, Dingqiang Chen, Yanmei Li, Guangchao Yu","doi":"10.1186\/1472-6750-14-8","country":"","institute":"","deparment":"","abstract":"Background: Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. As clinical MRSA diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for MRSA detection. This study aimed at developing a simple loop-mediated isothermal amplification (LAMP) assay targeting on orfX for the rapid detection of methicillin-resistance Staphylococcus aureus (MRSA).\n\nResults: The protocol was designed by targeting orfX, a highly conserved open reading frame in S. aureus. One hundred and sixteen reference strains, including 52 Gram-positive and 64 Gram-negative isolates, were included for evaluation and optimization of the orfX-LAMP assay. This assay had been further performed on 667 Staphylococcus (566 MRSA, 25 MSSA, 53 MRCNS and 23 MSCNS) strains and were comparatively validated by PCR assay using primers F3 and B3, with rapid template DNA processing, simple equipments (water bath) and direct result determination (both naked eye and under UV light) applied. The indispensability of each primer had been confirmed, and the optimal amplification was obtained under 65\u00b0C for 45 min. The 25 \u03bcl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be 10 DNA copies and 10 CFU\/reaction. High specificity was observed when orfX-LAMP assay was subjected to 116 reference strains. For application, 557 (98.4%, 557\/566) and 519 (91.7%, 519\/566) tested strains had been detected positive by LAMP and PCR assays. The detection rate, positive predictive value (PPV) and negative predictive value (NPV) of orfX-LAMP were 98.4%, 100% and 92.7% respectively.\n\nConclusions: The established orfX-LAMP assay had been demonstrated to be a valid and rapid detection method on MRSA.","lamp_id":[{"id":"LPB00348","pathogen":"Staphylococcus aureus","target":"orfX"}]},{"id":186,"pmid":34146608,"title":"A novel RdRp-based colorimetric RT-LAMP assay for rapid and sensitive detection of SARS-CoV-2 in clinical and sewage samples from Pakistan","year":2021,"journal":"Virus Research","authors":"Muhammad Farhan Ul Haque, Syeda Sadia Bukhari, Rabia Ejaz, Faheem Uz Zaman, Kamalalayam Rajan Sreejith, Naeem Rashid, Muhammad Umer, Naveed Shahzad","doi":"10.1016\/j.virusres.2021.198484","country":"Pakistan","institute":"University of the Punjab","deparment":"School of Biological Sciences","abstract":"Novel corona virus SARS-CoV-2, causing coronavirus disease 2019 (COVID-19), has become a global health challenge particularly for developing countries like Pakistan where overcrowded cities, inadequate sanitation, little health awareness and poor socioeconomic conditions exist. The SARS-CoV-2 has been known to spread primarily through direct contact and respiratory droplets. However, detection of SARS-CoV-2 in stool and sewage have raised the possibility of fecal-oral mode of transmission. Currently, quantitative reverse-transcriptase PCR (qRT-PCR) is the only method being used for SARS-CoV-2 detection, which requires expensive instrumentation, dedicated laboratory setup, highly skilled staff, and several hours to report results. Considering the high transmissibility and rapid spread, a robust, sensitive, specific and cheaper assay for rapid SARS-CoV-2 detection is highly needed. Herein, we report a novel colorimetric RT-LAMP assay for naked-eye detection of SARS-COV-2 in clinical as well as sewage samples. Our SARS-CoV-2 RdRp-based LAMP assay could successfully detect the virus RNA in 26\/28 (93%) of RT-PCR positive COVID-19 clinical samples with 100% specificity (n = 7) within 20 min. We also tested the effect of various additives on the performance of LAMP assay and found that addition of 1 mg\/ml bovine serum albumin (BSA) could increase the sensitivity of assay up to 10^1 copies of target sequence. Moreover, we also successfully applied this assay to detect SARS-CoV-2 in sewage waters collected from those areas of Lahore, a city of Punjab province of Pakistan, declared as virus hotspots by local government. Our optimized LAMP assay could provide a sensitive first tier strategy for SARS-CoV-2 screening and can potentially help diagnostic laboratories in better handling of high sample turnout during pandemic situation. By providing rapid naked-eye SARS-CoV-2 detection in sewage samples, this assay may support pandemic readiness and emergency response to any possible virus outbreaks in future.","lamp_id":[{"id":"LPB00350","pathogen":"SARS-CoV-2","target":"RdRp"}]},{"id":187,"pmid":34072209,"title":"Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis","year":2021,"journal":"International Journal of Molecular Sciences","authors":"Igor P. Oscorbin, Georgiy Yu. Shevelev, Ksenia A. Pronyaeva, Andrey A. Stepanov, Darya V. Shamovskaya, Olga V. Mishukova, Dmitrii V. Pyshnyi, Maksim L. Filipenko","doi":"10.3390\/ijms22115743","country":"Russia","institute":"Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS)","deparment":"Institute of Chemical Biology and Fundamental Medicine","abstract":"Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification that is more stable and resistant to DNA amplification inhibitors than conventional PCR. LAMP multiplexing with reverse transcription allows for the single-tube amplification of several RNA fragments, including an internal control sample, which provides the option of controlling all analytical steps. We developed a method of SARS-CoV-2 viral RNA detection based on multiplex reverse-transcription LAMP, with single-tube qualitative analysis of SARS-CoV-2 RNA and MS2 phage used as a control RNA. The multiplexing is based on the differences in characteristic melting peaks generated during the amplification process. The developed technique detects at least 20 copies of SARS-CoV-2 RNA per reaction on a background of 12,000 MS2 RNA copies. The total time of analysis does not exceed 40 min. The method validation, performed on 125 clinical samples of patients' nasal swabs, showed a 97.6% concordance rate with the results of real-time (RT)-PCR assays. The developed multiplexed LAMP can be employed as an alternative to PCR in diagnostic practice to save personnel and equipment time.","lamp_id":[{"id":"LPB00351","pathogen":"SARS-CoV-2","target":"E"}]},{"id":188,"pmid":31456171,"title":"CelB is a suitable marker for rapid and specific identification of Klebsiella pneumoniae by the loop-mediated isothermal amplification (LAMP) assay","year":2019,"journal":"Brazilian Journal of Microbiology","authors":"Yichen Tian, Lefei Wang, Jinyang Zhang, Qinqin Han, Xue-Shan Xia, Yuzhu Song, Guangying Yang","doi":"10.1007\/s42770-019-00144-9","country":"","institute":"","deparment":"","abstract":"Klebsiella pneumoniae belongs to Enterobacteriaceae, which is the commonest bacterium causing nosocomial respiratory tract infection. It ranks second in bacteremia and urinary tract infection in gram-negative bacteria. Therefore, the rapid and accurate identification of K. pneumoniae was of great significance for the guide of clinical medication, and timely treatment of patients. The purpose of this study was to establish a rapid and sensitive molecular detection method for K. pneumoniae based on loop-mediated isothermal amplification (LAMP) technology. Firstly, local BLAST and NCBI BLAST were used to analyze the genome of K. pneumoniae. According to the principle of interspecific and intraspecific specificity, CelB (GenBank ID 11847805) was selected as the specific gene. Then, the LAMP and PCR identification systems were established with this target gene. Thirty-six clinical isolates of K. pneumoniae and 50 non-K. pneumoniae were used for the specific evaluation, and both LAMP and PCR could specifically distinguish K. pneumoniae from non-K. pneumoniae. A 10-fold series diluted positive plasmids and simulated infected blood samples were used as the templates in the sensitivity assay, and the results showed that the sensitivity could reach 1 copy\/reaction. In summary, a rapid, specific, and sensitive LAMP method was established to detect K. pneumoniae in clinics.","lamp_id":[{"id":"LPB00352","pathogen":"Klebsiella pneumoniae subsp. pneumoniae HS11286","target":"CelB"}]},{"id":189,"pmid":34264402,"title":"Development of a loop-mediated isothermal amplification assay for the rapid detection of six common respiratory viruses","year":2021,"journal":"European Journal of Clinical Microbiology & Infectious Diseases","authors":"Nianzhen Chen, Yuying Si, Gen Li, Ming Zong, Wenyan Zhang, Yangqin Ye, Lieying Fan","doi":"10.1007\/s10096-021-04300-8","country":"People's Republic of China","institute":"Shanghai East Hospital","deparment":"Department of Clinical Laboratory","abstract":"Due to the highly contagious and spreads quickly of respiratory infectious diseases (ADR), the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Here, we develop an approach based on loop-mediated isothermal amplification (LAMP) for the detection of influenza A virus (Flu A), Flu A subtypes H1N1and H3N2, influenza B virus (Flu B), respiratory syncytial virus (RSV) subtypes A and B, human adenovirus (HAdV), parainfluenza virus (PIV) subtypes 1 and 3, and human rhinovirus (HRV) simultaneously. We designed primers specific to detect respiratory viruses above, optimized the RT-LAMP assay and evaluated it for its sensitivity and specificity of detection using real-time monitoring based on SYBR Green I. We also evaluated the result of our RT-LAMP assay on 638 nasopharyngeal swab specimens with the commercial RT-PCR by Cohen's Kappa. The inconsistent results were verified by Sanger sequencing furtherly. The developed RT-LAMP assay displayed a detection limit of 1 \u00d7 10^2 copies\/ml RNA close to that of RT-PCR; no cross-reactivity was observed in the 10 kinds of viruses studied. The results obtained with 638 clinical samples indicate that the developed method has high specificity (0.988-1) and sensitivity (0.863-1) for viruses studied, and the Kappa value of all viruses was more than 0.85 revealed an excellent agreement between the two methods. We developed an RT-LAMP-based method and optimized for the detection of common respiratory viruses. It may be a powerful tool for rapid and reliable clinical diagnosis of ADR in primary hospitals.","lamp_id":[{"id":"LPB00355","pathogen":"Influenza A virus","target":"M"},{"id":"LPB00356","pathogen":"Influenza A virus (H1N1)","target":"M"},{"id":"LPB00357","pathogen":"Influenza A virus (H3N2)","target":"HA"},{"id":"LPB00358","pathogen":"Influenza B virus","target":"NS1"},{"id":"LPB00359","pathogen":"HRSV A","target":"Polymerase"},{"id":"LPB00360","pathogen":"HRSV B","target":"Fusion glycoprotein"},{"id":"LPB00361","pathogen":"HAdV","target":"Hexon"},{"id":"LPB00362","pathogen":"HPIV-1","target":"HA"},{"id":"LPB00363","pathogen":"HPIV-3","target":"HA"},{"id":"LPB00364","pathogen":"HRV","target":"Polyprotein"}]},{"id":190,"pmid":34248284,"title":"Sensitive and rapid on-site detection of SARS-CoV-2 using a gold nanoparticle-based high-throughput platform coupled with CRISPR\/Cas12-assisted RT-LAMP","year":2021,"journal":"Sensors and Actuators B: Chemical","authors":"Yaqin Zhang, Minyan Chen, Chengrong Liu, Jiaqi Chen, Xinyi Luo, Yingying Xue, Qiming Liang, Li Zhou, Yu Tao, Mingqiang Li, Di Wang, Jianhua Zhou, Jiasi Wang","doi":"10.1016\/j.snb.2021.130411","country":"China","institute":"Jilin University","deparment":"School of Life Sciences","abstract":"The outbreak of corona virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic. The high infectivity of SARS-CoV-2 highlights the need for sensitive, rapid and on-site diagnostic assays of SARS-CoV-2 with high-throughput testing capability for large-scale population screening. The current detection methods in clinical application need to operate in centralized labs. Though some on-site detection methods have been developed, few tests could be performed for high-throughput analysis. We here developed a gold nanoparticle-based visual assay that combines with CRISPR\/Cas12a-assisted RT-LAMP, which is called Cas12a-assisted RT-LAMP\/AuNP (CLAP) assay for rapid and sensitive detection of SARS-CoV-2. In optimal condition, we could detect down to 4 copies\/\u03bcL of SARS-CoV-2 RNA in 40 min. by naked eye. The sequence-specific recognition character of CRISPR\/Cas12a enables CLAP a superior specificity. More importantly, the CLAP is easy for operation that can be extended to high-throughput test by using a common microplate reader. The CLAP assay holds a great potential to be applied in airports, railway stations, or low-resource settings for screening of suspected people. To the best of our knowledge, this is the first AuNP-based colorimetric assay coupled with Cas12 and RT-LAMP for on-site diagnosis of COVID-19. We expect CLAP assay will improve the current COVID-19 screening efforts, and make contribution for control and mitigation of the pandemic.","lamp_id":[{"id":"LPB00365","pathogen":"SARS-CoV-2","target":"N"}]},{"id":191,"pmid":34169944,"title":"CRISPR\/Cas12a-mediated gold nanoparticle aggregation for colorimetric detection of SARS-CoV-2","year":2021,"journal":"Chemical Communications","authors":"Yiren Cao, Jinjun Wu, Bo Pang, Hongquan Zhang, X Chris Le","doi":"10.1039\/d1cc02546e","country":"Canada","institute":"University of Alberta","deparment":"Department of Laboratory Medicine and Pathology","abstract":"The trans-cleavage activity of the target-activated CRISPR\/Cas12a liberated an RNA crosslinker from a molecular transducer, which facilitated the assembly of gold nanoparticles. Integration of the molecular transducer with isothermal amplification and CRISPR\/Cas12a resulted in visual detection of the N gene and E gene of SARS-CoV-2 in 45 min.","lamp_id":[{"id":"LPB00366","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00367","pathogen":"SARS-CoV-2","target":"E"}]},{"id":192,"pmid":34336708,"title":"Rapid and Visual Detection of SARS-CoV-2 Using Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification Linked With Gold Nanoparticle-Based Lateral Flow Biosensor","year":2021,"journal":"Frontiers in Cellular and Infection Microbiology","authors":"Xu Che, Qingxue Zhou, Shijun Li, Hao Yan, Bingcheng Chang, Yuexia Wang, Shilei Dong","doi":"10.3389\/fcimb.2021.581239","country":"China","institute":"Guizhou University of Traditional Chinese Medicine\/Guizhou Provincial Centre for Disease Control and Prevention","deparment":"The Second Clinical College\/Central Laboratory of the Second Affiliated Hospital\/Laboratory of Bacterial Infectious Disease of Experimental Centre","abstract":"Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has caused the outbreak of coronavirus disease 2019 (COVID-19) all over the world. In the absence of appropriate antiviral drugs or vaccines, developing a simple, rapid, and reliable assay for SARS-CoV-2 is necessary for the prevention and control of the COVID-19 transmission.\n\nMethods: A novel molecular diagnosis technique, named multiplex reverse transcription loop-mediated isothermal amplification, that has been linked to a nanoparticle-based lateral flow biosensor (mRT-LAMP-LFB) was applied to detect SARS-CoV-2 based on the SARS-CoV-2 RdRp and N genes, and the mRT-LAMP products were analyzed using nanoparticle-based lateral flow biosensor. The mRT-LAMP-LFB amplification conditions, including the target RNA concentration, amplification temperature, and time were optimized. The sensitivity and specificity of the mRT-LAMP-LFB method were tested in the current study, and the mRT-LAMP-LFB assay was applied to detect the SARS-CoV-2 virus from clinical samples and artificial sputum samples.\n\nResults: The SARS-CoV-2 specific primers based on the RdRp and N genes were valid for the establishment of mRT-LAMP-LFB assay to detect the SARS-CoV-2 virus. The multiple-RT-LAMP amplification condition was optimized at 63\u00b0C for 30 min. The full process, including reaction preparation, viral RNA extraction, RT-LAMP, and product identification, could be achieved in 80 min. The limit of detection (LoD) of the mRT-LAMP-LFB technology was 20 copies per reaction. The specificity of mRT-LAMP-LFB detection was 100%, and no cross-reactions to other respiratory pathogens were observed.\n\nConclusion: The mRT-LAMP-LFB technique developed in the current study is a simple, rapid, and reliable method with great specificity and sensitivity when it comes to identifying SARS-CoV-2 virus for prevention and control of the COVID-19 disease, especially in resource-constrained regions of the world.","lamp_id":[{"id":"LPB00368","pathogen":"SARS-CoV-2","target":"RdRp"},{"id":"LPB00369","pathogen":"SARS-CoV-2","target":"N"}]},{"id":193,"pmid":34268131,"title":"A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2","year":2021,"journal":"Frontiers in Cellular and Infection Microbiology","authors":"Eduardo Juscamayta-L\u00f3pez, Faviola Valdivia, Helen Horna, David Tarazona, Liza Linares, Nancy Rojas, Maribel Huaringa","doi":"10.3389\/fcimb.2021.653616","country":"Peru","institute":"Instituto Nacional de Salud","deparment":"Laboratorio de Infecciones Respiratorias Agudas","abstract":"Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major threat to public health. Rapid molecular testing for convenient and timely diagnosis of SARS-CoV-2 infections represents a challenge that could help to control the current pandemic and prevent future outbreaks. We aimed to develop and validate a multiplex and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using lyophilized LAMP reagents for sensitive and rapid detection of SARS-CoV-2. LAMP primers were designed for a set of gene targets identified by a genome-wide comparison of viruses. Primer sets that showed optimal features were combined into a multiplex RT-LAMP assay. Analytical validation included assessment of the limit of detection (LoD), intra- and inter-assay precision, and cross-reaction with other respiratory pathogens. Clinical performance compared to that of real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was assessed using 278 clinical RNA samples isolated from swabs collected from individuals tested for COVID-19. The RT-LAMP assay targeting the RNA-dependent RNA polymerase (RdRp), membrane (M), and ORF1ab genes achieved a comparable LoD (0.65 PFU\/mL, CT=34.12) to RT-qPCR and was 10-fold more sensitive than RT-qPCR at detecting viral RNA in clinical samples. Cross-reactivity to other respiratory pathogens was not observed. The multiplex RT-LAMP assay demonstrated a strong robustness and acceptable intra- and inter-assay precision (mean coefficient of variation, 4.75% and 8.30%). Diagnostic sensitivity and specificity values were 100.0% (95% CI: 97.4-100.0%) and 98.6% (95% CI: 94.9-99.8%), respectively, showing high consistency (Cohen's kappa, 0.986; 95% CI: 0.966-1.000; p<0.0001) compared to RT-qPCR. The novel one-step multiplex RT-LAMP assay is storable at room temperature and showed similar diagnostic accuracy to conventional RT-qPCR, while being faster (<45 min), simpler, and cheaper. The new assay could allow reliable and early diagnosis of SARS-CoV-2 infections in primary health care. It may aid large-scale testing in resource-limited settings, especially if it is integrated into a point-of-care diagnostic device.","lamp_id":[{"id":"LPB00370","pathogen":"SARS-CoV-2","target":"RdRp"},{"id":"LPB00374","pathogen":"SARS-CoV-2","target":"M"},{"id":"LPB00380","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":194,"pmid":34711332,"title":"Capillary-based reverse transcriptase loop-mediated isothermal amplification for cost-effective and rapid point-of-care COVID-19 testing","year":2021,"journal":"Analytica Chimica Acta","authors":"Anto J U K John, Peijun J W He, Ioannis N Katis, P P Galanis, Alice H Iles, Robert W Eason, Collin L Sones","doi":"10.1016\/j.aca.2021.339002","country":"United Kingdom","institute":"University of Southampton","deparment":"Optoelectronics Research Centre","abstract":"As the SARS-CoV-2 pandemic continues to spread, the necessity for rapid, easy diagnostic capabilities could never have been more crucial. With this aim in mind, we have developed a cost-effective and time-saving testing methodology\/strategy that implements a sensitive reverse transcriptase loop-mediated amplification (RT-LAMP) assay within narrow, commercially available and cheap, glass capillaries for detection of the SARS-CoV-2 viral RNA. The methodology is compatible with widely used laboratory-based molecular testing protocols and currently available infrastructure. It employs a simple rapid extraction protocol that lyses the virus, releasing sufficient genetic material for amplification. This extracted viral RNA is then amplified using a SARS-CoV-2 RT-LAMP kit, at a constant temperature and the resulting amplified product produces a colour change which can be visually interpreted. This testing protocol, in conjunction with the RT-LAMP assay, has a sensitivity of \u223c100 viral copies per reaction of a sample and provides results in a little over 30 min. As the assay is carried out in a water bath, commonly available within most testing laboratories, it eliminates the need for specialised instruments and associated skills. In addition, our testing pathway requires a significantly reduced quantity of reagents per test while providing comparable sensitivity and specificity to the RT-LAMP kit used in this study. While the conventional technique requires 25 \u03bcl of reagent, our test only utilises less than half the quantity (10 \u03bcl). Thus, with its minimalistic approach, this capillary-based assay could be a promising alternative to the conventional testing, owing to the fact that it can be performed in resource-limited settings, using readily available apparatus, and has the potential of increasing the overall testing capacity, while also reducing the burden on supply chains for mass testing.","lamp_id":[{"id":"LPB00371","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00372","pathogen":"SARS-CoV-2","target":"N"}]},{"id":195,"pmid":35129304,"title":"Plasmonic LAMP: Improving the Detection Specificity and Sensitivity for SARS-CoV-2 by Plasmonic Sensing of Isothermally Amplified Nucleic Acids","year":2022,"journal":"Small","authors":"Haihang Ye, Chance Nowak, Yaning Liu, Yi Li, Tingting Zhang, Leonidas Bleris, Zhenpeng Qin","doi":"10.1002\/smll.202107832","country":"USA","institute":"The University of Texas at Dallas","deparment":"Department of Mechanical Engineering","abstract":"The ability to detect pathogens specifically and sensitively is critical to combat infectious diseases outbreaks and pandemics. Colorimetric assays involving loop-mediated isothermal amplification (LAMP) provide simple readouts yet suffer from the intrinsic non-template amplification. Herein, a highly specific and sensitive assay relying on plasmonic sensing of LAMP amplicons via DNA hybridization, termed as plasmonic LAMP, is developed for the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) RNA detection. This work has two important advances. First, gold and silver (Au-Ag) alloy nanoshells are developed as plasmonic sensors that have 4-times stronger extinction in the visible wavelengths and give a 20-times lower detection limit for oligonucleotides over Au counterparts. Second, the integrated method allows cutting the complex LAMP amplicons into short repeats that are amendable for hybridization with oligonucleotide-functionalized Au-Ag nanoshells. In the SARS-CoV-2 RNA detection, plasmonic LAMP takes \u224875 min assay time, achieves a detection limit of 10 copies per reaction, and eliminates the contamination from non-template amplification. It also shows better detection specificity and sensitivity over commercially available LAMP kits due to the additional sequence identification. This work opens a new route for LAMP amplicon detection and provides a method for virus testing at its early representation.","lamp_id":[{"id":"LPB00373","pathogen":"SARS-CoV-2","target":"N"}]},{"id":196,"pmid":21621554,"title":"Visual detection of human enterovirus 71 subgenotype C4 and Coxsackievirus A16 by reverse transcription loop-mediated isothermal amplification with the hydroxynaphthol blue dye","year":2011,"journal":"Journal of Virological Methods","authors":"Kai Nie, Yong Zhang, Le Luo, Meng-Jie Yang, Xiu-Mei Hu, Miao Wang, Shuang-Li Zhu, Feng Han, Wen-Bo Xu, Xue-Jun Ma","doi":"10.1016\/j.jviromet.2011.05.020","country":"China","institute":"National Institute for Viral Disease Control and Prevention","deparment":"State Key Laboratory for Molecular Virology and Genetic Engineering","abstract":"A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of human enterovirus 71 subgenotype C4 (EV71-C4) and Coxsackievirus A16 (CVA16) infection, respectively. The reaction was performed in one step in a single tube at 65\u00b0C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limits of the RT-LAMP assay were 0.33 and 1.58 of a 50% tissue culture infective dose (TCID(50)) per reaction based on 10-fold dilutions of a titrated EV71 or CVA16 strain, respectively. No cross-reaction was observed with Coxsackievirus A (CVA) viruses (CVA2, 4, 5, 7, 9, 10, 14, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3, 6, 11, and 19). The assay was further evaluated with 47 clinical stool specimens diagnosed previously with EV71, CVA16 or other human enterovirus infections. Virus isolates from stool samples were confirmed by virus neutralization testing and sequencing. RT-LAMP with HNB dye was demonstrated to be a sensitive and cost-effective assay for rapid visual detection of human EV71-C4 and CVA16.","lamp_id":[{"id":"LPB00375","pathogen":"EV-A71","target":"VP1"},{"id":"LPB00376","pathogen":"CVA16","target":"VP1"}]},{"id":197,"pmid":23862439,"title":"A closed-tube detection of loop-mediated isothermal amplification (LAMP) products using a wax-sealed fluorescent intercalator","year":2013,"journal":"Journal of Nanoscience and Nanotechnology","authors":"Chao Liang, Sijia Cheng, Yanan Chu, Haiping Wu, Binjie Zou, Huan Huang, Tao Xi, Guohua Zhou","doi":"10.1166\/jnn.2013.6497","country":"China","institute":"China Pharmaceutical University","deparment":"College of Life Science and Technology","abstract":"LAMP is an isothermal amplification method that can achieve ultra-high sensitivity and specificity. However, the conventional detection of LAMP amplicons can lead to cross-contamination due to the need to open the reaction tube which contains a large number of amplicons. To achieve closed-tube LAMP detection, we have developed a method that separates a solution of SYBR Green I (SGI) from the LAMP reagents using temperature-sensitive wax. The SGI is sealed in the bottom of the tube so not to interfere with the LAMP reaction, but is released into the mixture after the completion of the reaction by melting the wax. To enable the analysis of the closed-tube LAMP samples automatically, an instrument based on this new method was constructed. The background measurement of the LAMP due to primer dimers was significantly reduced by detecting the amplicons at 75 degrees C. HBV and 2009 H1N1 virus were successfully analyzed by the LAMP assay using tubes containing wax-sealed SGI and the prototype instrument, indicating that the method has the advantage of easy set-up (no extra components need to be added into the LAMP mixture for detection), high sensitivity (fluorescent intercalator), low background (detected at 75 degrees C) and no cross-contamination (closed-tube). Therefore, the novel LAMP detection, coupled with the instrument has the potential to be a diagnostic tool for a number of clinical applications in hospitals as well as on-site screening of pathogenic agents.","lamp_id":[{"id":"LPB00377","pathogen":"Influenza A virus (H1N1)","target":null}]},{"id":198,"pmid":23272248,"title":"Evaluation of a direct reverse transcription loop-mediated isothermal amplification method without RNA extraction for the detection of human enterovirus 71 subgenotype C4 in nasopharyngeal swab specimens","year":2012,"journal":"PLoS One","authors":"Kai Nie, Shun-Xiang Qi, Yong Zhang, Le Luo, Yun Xie, Meng-Jie Yang, Yi Zhang, Jin Li, Hongwei Shen, Qi Li, Xue-Jun Ma","doi":"10.1371\/journal.pone.0052486","country":"People's Republic of China","institute":"National Institute for Viral Disease Control and Prevention","deparment":"State Key Laboratory for Molecular Virology and Genetic Engineering","abstract":"Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD) worldwide and has been associated with neurological complications which resulted in fatalities during recent outbreak in Asia pacific region. A direct reverse transcription loop-mediated isothermal amplification (direct RT-LAMP) assay using heat-treated samples without RNA extraction was developed and evaluated for the detection of EV71 subgenotype C4 in nasopharyngeal swab specimens. The analytical sensitivity and specificity of the direct RT-LAMP assay were examined. The detection limit of the direct RT-LAMP assays was 1.6 of a 50% tissue culture infective dose (TCID(50)) per reaction and no cross-reaction was observed with control viruses including Cosackievirus A (CVA) viruses (CVA2,4,5,7,9,10,14,16, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3,6,11, and 19). The direct RT-LAMP assay was evaluated and compared to both RT-LAMP and quantitative real-time PCR (qRT-PCR) in detecting EV71 infection with 145 nasopharyngeal swab specimens. The clinical performance demonstrated the sensitivity and specificity of direct RT-LAMP was reported to be 90.3% and 100% respectively, compared to RT-LAMP, and 86.83% and 100% respectively, compared to qRT-PCR. These data demonstrated that the direct RT-LAMP assay can potentially be developed for the point of care screening of EV71 infection in China.","lamp_id":[{"id":"LPB00378","pathogen":"EV-A71","target":"VP1"}]},{"id":199,"pmid":22838725,"title":"Visual detection of the human metapneumovirus using reverse transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye","year":2012,"journal":"Virology Journal","authors":"Xiang Wang, Qian Zhang, Fang Zhang, Fenlian Ma, Wenzhi Zheng, Zhihui Zhao, Yinglong Bai, Lishu Zheng","doi":"10.1186\/1743-422X-9-138","country":"People's Republic of China","institute":"National Institute for Viral Disease Control and Prevention","deparment":"State Key Laboratory for Molecular Virology and Genetic Engineering","abstract":"Background: Human metapneumovirus (hMPV) is a major cause of acute respiratory infections ranging from wheezing to bronchiolitis and pneumonia in children worldwide. The objective of this study is to develop a visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of hMPV and applied to the clinical samples.\n\nResults: In this study, visual RT-LAMP assay for hMPV was performed in one step with the addition of hydroxynaphthol blue (HNB), and were used to detect respiratory samples. Six primers, including two outer primers (F3 and B3), two inner primers (FIP, BIP) and two loop primers (LF and LB), were designed for hMPV N gene by the online software. Moreover, the RT-LAMP assay showed good specificity and no cross-reactivity was observed with human rhinovirus (HRV), human respiratory syncytial Virus (RSV), or influenza virus A\/PR\/8\/34 (H1N1). The detection limit of the RT-LAMP assay was approximately ten viral RNA copies, lower than that of traditional reverse transcriptase polymerase chain reaction (RT-PCR) 100 RNA copies. In the 176 nasopharyngeal samples, 23 (13.1%) were conformed as hMPV positive by RT-LAMP, but 18 (10.2%) positive by RT-PCR.\n\nConclusion: Compared with conventional RT-PCR, the visual hMPV RT-LAMP assay performed well in the aspect of detect time, sensitivity, specificity and visibility. It is anticipated that the RT-LAMP will be used for clinical tests in hospital or field testing during outbreaks and in emergency.","lamp_id":[{"id":"LPB00379","pathogen":"HMPV","target":"N"}]},{"id":200,"pmid":24713896,"title":"Detection of enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification","year":2014,"journal":"Indian Journal of Medical Microbiology","authors":"D Wang, X Wang, Y Geng, C An","doi":"10.4103\/0255-0857.129779","country":"China","institute":"China Medical University","deparment":"Department of Medical Microbiology and Parasitolog","abstract":"Purpose: The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD) for an early treatment by using loop-mediated isothermal amplification (LAMP) technique.\n\nMaterials and methods: A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for detecting EV71 virus was developed, the specificity and sensitivity of RT-LAMP was tested, and the clinical specimens was assayed by the RT-LAMP comparing with conventional reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR.\n\nResults: A total of 116 clinical specimens from the suspected HFMD individual were detected with the RT-LAMP. The detection rate for EV71 was 56.89% by RT-LAMP, 41.38% by real-time PCR and 34.48% by RT-PCR. The minimum detection limit of RT-LAMP was 0.01 PFU, both of RT-PCR and real-time PCR was 0.1PFU. Non-cross-reactive amplification with other enteroviruses was detected in the survey reports.\n\nConclusions: The effectiveness of RT-LAMP is higher than RT-PCR and real-time PCR. The protocol is easy to operate and time saving. It was not an expensive instrument, which was needed; it is an applicable method for rapid diagnosis of the disease, especially in resource-poor countries or in developing countries.","lamp_id":[{"id":"LPB00381","pathogen":"EV-A71","target":"VP1"}]},{"id":201,"pmid":34262053,"title":"Oil Immersed Lossless Total Analysis System for Integrated RNA Extraction and Detection of SARS-CoV-2","year":2021,"journal":"Nature Communications","authors":"Duane S. Juang, Terry D. Juang, Dawn M. Dudley, Christina M. Newman, Molly A. Accola, William M. Rehrauer, Thomas C. Friedrich, David H. O'Connor, David J. Beebe","doi":"10.1038\/s41467-021-24463-4","country":"USA","institute":"University of Wisconsin-Madison","deparment":"Department of Biomedical Engineering","abstract":"The COVID-19 pandemic exposed difficulties in scaling current quantitative PCR (qPCR)-based diagnostic methodologies for large-scale infectious disease testing. Bottlenecks include lengthy multi-step processes for nucleic acid extraction followed by qPCR readouts, which require costly instrumentation and infrastructure, as well as reagent and plastic consumable shortages stemming from supply chain constraints. Here we report an Oil Immersed Lossless Total Analysis System (OIL-TAS), which integrates RNA extraction and detection onto a single device that is simple, rapid, cost effective, and requires minimal supplies and infrastructure to perform. We validated the performance of OIL-TAS using contrived SARS-CoV-2 viral particle samples and clinical nasopharyngeal swab samples. OIL-TAS showed a 93% positive predictive agreement (n = 57) and 100% negative predictive agreement (n = 10) with clinical SARS-CoV-2 qPCR assays in testing clinical samples, highlighting its potential to be a faster, cheaper, and easier-to-deploy alternative for infectious disease testing.","lamp_id":[{"id":"LPB00382","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00383","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":202,"pmid":34307953,"title":"Simultaneous detection of SARS-CoV-2 and pandemic (H1N1) 2009 virus with real-time isothermal platform","year":2021,"journal":"Heliyon","authors":"Lin Yu, Jingyao Wang, Xuelong Li, Lingling Mao, Yi Sui, Weihua Chen, Vicent Pelechano, Xing Guo, Xiushan Yin","doi":"10.1016\/j.heliyon.2021.e07584","country":"China","institute":"Shenyang University of Chemical Technology","deparment":"Applied Biology Laboratory","abstract":"The recent ongoing outbreak of novel coronavirus SARS-CoV-2 (known as COVID-19) is a severe threat to human health worldwide. By press time, more than 3.3 million people have died from COVID-19, with many countries experiencing peaks in infections and hospitalizations. The main symptoms of infection with SARS-CoV-2 include fever, chills, coughing, shortness of breath or difficulty breathing, fatigue, muscle or body aches and pains. While the symptoms of the pandemic (H1N1) 2009 virus have many similarities to the signs and transmission routes of the novel coronavirus, e.g., fever, cough, sore throat, body aches, headache, chills and fatigue. And a few cases of serious illness, rapid progress, can appear viral pneumonia, combined with respiratory failure, multiple organ function damage, serious people can die. Therefore, there is an urgent need to develop a rapid and accurate field diagnostic method to effectively identify the two viruses and treat these early infections on time, thus helping to control the spread of the disease. Among molecular detection methods, RT-LAMP (real-time reverse transcription-loop-mediated isothermal amplification) has some advantages in pathogen detection due to its rapid, accurate and effective detection characteristics. Here, we combined the primers of the two viruses with the fluorescent probes on the RT-LAMP detection platform to detect the two viruses simultaneously. Firstly, RT-LAMP method was used respectively to detect the two viruses at different concentrations to determine the effectiveness and sensitivity of probe primers to the RNA samples. And then, the two virus samples were detected simultaneously in the same reaction tube to validate if testing for the two viruses together had an impact on the results compared to detecting alone. We verified the detection efficiency of three highly active BST variants during RT-LAMP assay. We expect that this assay can effectively and accurately distinguish COVID-19 from the pandemic (H1N1) 2009, so that these two diseases with similar symptoms can be appropriately differentiated and treated.","lamp_id":[{"id":"LPB00384","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00385","pathogen":"Influenza A virus (H1N1)","target":"HA"}]},{"id":203,"pmid":34312441,"title":"An ultra-portable, self-contained point-of-care nucleic acid amplification test for diagnosis of active COVID-19 infection","year":2021,"journal":"Scientific Reports","authors":"Hao Deng, Asanka Jayawardena, Jianxiong Chan, Sher Maine Tan, Tuncay Alan, Patrick Kwan","doi":"10.1038\/s41598-021-94652-0","country":"Australia","institute":"Monash University","deparment":"Department of Mechanical and Aerospace Engineering\/Department of Neuroscience","abstract":"There is currently a high level of demand for rapid COVID-19 tests, that can detect the onset of the disease at point of care settings. We have developed an ultra-portable, self-contained, point-of-care nucleic acid amplification test for diagnosis of active COVID-19 infection, based on the principle of loop mediated isothermal amplification (LAMP). The LAMP assay is 100% sensitive and specific to detect a minimum of 300 RNA copies\/reaction of SARS-CoV-2. All of the required sample transportation, lysing and amplification steps are performed in a standalone disposable cartridge, which is controlled by a battery operated, pocket size (6x9x4cm3) unit. The test is easy to operate and does not require skilled personnel. The total time from sample to answer is approximately 35 min; a colorimetric readout indicates positive or negative results. This portable diagnostic platform has significant potential for rapid and effective testing in community settings. This will accelerate clinical decision making, in terms of effective triage and timely therapeutic and infection control interventions.","lamp_id":[{"id":"LPB00386","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00388","pathogen":"SARS-CoV-2","target":"E"}]},{"id":204,"pmid":33717251,"title":"Evaluation of loop-mediated isothermal amplification assays for rapid detection of bla KPC producing Serratia spp. in clinical specimens: A prospective diagnostic accuracy study","year":2021,"journal":"Experimental and Therapeutic Medicine","authors":"Xinwei Liu, Dayang Zou, Chunxia Wang, Xiaoqian Zhang, Dongxu Pei, Wei Liu, Yongwei Li","doi":"10.3892\/etm.2021.9739","country":"","institute":"","deparment":"","abstract":"The prevalence of carbapenem-resistant Serratia spp. is increasing owing to the propagation of \u03b2 lactamase Klebsiella pneumoniae carbapenemase (blaKPC) and it has become one of the major global health concerns. As effective therapies for such resistant pathogens are limited, there is a great need for the rapid and sensitive characterization of the pathogen. In the present study, a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Serratia spp. with blaKPC in pure cultures and clinical specimens was developed. A calcein indicator and real-time turbidity recording system were used to assess the LAMP reaction. The LAMP assay was compared with conventional PCR and real-time PCR kits for the target pathogen. The desired amplification was achieved using selected primers and detection was possible using both the calcein indicator method and the real-time turbity recording system at 65\u02daC for 60 min. The sensitivity of the detection system for blaKPC-producing Serratia spp. reached a detection limit of 3.92 pg\/\u00b5l DNA, which was 10 times more sensitive than conventional PCR. Specificity testing indicated that the primers were highly specific. Compared with conventional culture methods and real-time PCR, the LAMP assay was more sensitive, easier for laboratory staff to master and less influenced by the clinical specimen matrix. In conclusion, a LAMP assay for blaKPC-producing Serratia spp. that permitted rapid, sensitive and economical detection for this pathogen was successfully developed. Comparisons with alternative methods indicated that the LAMP assay was more feasible in a clinical setting.","lamp_id":[{"id":"LPB00387","pathogen":"Serratia marcescens","target":"luxS"},{"id":"LPB00389","pathogen":"Klebsiella pneumoniae","target":"blaKPC"}]},{"id":205,"pmid":34114589,"title":"Sample-to-answer COVID-19 nucleic acid testing using a low-cost centrifugal microfluidic platform with bead-based signal enhancement and smartphone read-out","year":2021,"journal":"Lab on a Chip","authors":"Ruben R G Soares, Ahmad S Akhtar, In\u00eas F Pinto, Noa Lapins, Donal Barrett, Gustaf Sandh, Xiushan Yin, Vicent Pelechano, Aman Russom","doi":"10.1039\/d1lc00266j","country":"Sweden","institute":"KTH Royal Institute of Technology","deparment":"Department of Protein Science","abstract":"With its origin estimated around December 2019 in Wuhan, China, the ongoing SARS-CoV-2 pandemic is a major global health challenge. The demand for scalable, rapid and sensitive viral diagnostics is thus particularly pressing at present to help contain the rapid spread of infection and prevent overwhelming the capacity of health systems. While high-income countries have managed to rapidly expand diagnostic capacities, such is not the case in resource-limited settings of low- to medium-income countries. Aiming at developing cost-effective viral load detection systems for point-of-care COVID-19 diagnostics in resource-limited and resource-rich settings alike, we report the development of an integrated modular centrifugal microfluidic platform to perform loop-mediated isothermal amplification (LAMP) of viral RNA directly from heat-inactivated nasopharyngeal swab samples. The discs were pre-packed with dried n-benzyl-n-methylethanolamine modified agarose beads used to selectively remove primer dimers, inactivate the reaction post-amplification and allowing enhanced fluorescence detection via a smartphone camera. Sample-to-answer analysis within 1 hour from sample collection and a detection limit of approximately 100 RNA copies in 10 \u03bcL reaction volume were achieved. The platform was validated with a panel of 162 nasopharyngeal swab samples collected from patients with COVID-19 symptoms, providing a sensitivity of 96.6% (82.2-99.9%, 95% CI) for samples with Ct values below 26 and a specificity of 100% (90-100%, 95% CI), thus being fit-for-purpose to diagnose patients with a high risk of viral transmission. These results show significant promise towards bringing routine point-of-care COVID-19 diagnostics to resource-limited settings.","lamp_id":[{"id":"LPB00390","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00391","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":206,"pmid":34529736,"title":"WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva","year":2021,"journal":"PLoS One","authors":"David Ng, Ana Pinharanda, Merly C Vogt, Ashok Litwin-Kumar, Kyle Stearns, Urvashi Thopte, Enrico Cannavo, Armen Enikolopov, Felix Fiederling, Stylianos Kosmidis, Barbara Noro, Ines Rodrigues-Vaz, Hani Shayya, Peter Andolfatto, Darcy S Peterka, Tanya Tabachnik, Jeanine D'Armiento, Monica Goldklang, Andres Bendesky","doi":"10.1371\/journal.pone.0257464","country":"USA","institute":"Columbia University","deparment":"Zuckerman Mind Brain Behavior Institute","abstract":"Despite the development of effective vaccines against SARS-CoV-2, epidemiological control of the virus is still challenging due to slow vaccine rollouts, incomplete vaccine protection to current and emerging variants, and unwillingness to get vaccinated. Therefore, frequent testing of individuals to identify early SARS-CoV-2 infections, contact-tracing and isolation strategies remain crucial to mitigate viral spread. Here, we describe WHotLAMP, a rapid molecular test to detect SARS-CoV-2 in saliva. WHotLAMP is simple to use, highly sensitive (~4 viral particles per microliter of saliva) and specific, as well as inexpensive, making it ideal for frequent screening. Moreover, WHotLAMP does not require toxic chemicals or specialized equipment and thus can be performed in point-of-care settings, and may also be adapted for resource-limited environments or home use. While applied here to SARS-CoV-2, WHotLAMP can be modified to detect other pathogens, making it adaptable for other diagnostic assays, including for use in future outbreaks.","lamp_id":[{"id":"LPB00392","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":207,"pmid":34546709,"title":"A Novel Miniature CRISPR-Cas13 System for SARS-CoV-2 Diagnostics","year":2021,"journal":"ACS Synthetic Biology","authors":"Ahmed Mahas, Qiaochu Wang, Tin Marsic, Magdy M. Mahfouz","doi":"10.1021\/acssynbio.1c00181","country":"","institute":"","deparment":"","abstract":"Rapid, point-of-care (POC) diagnostics are essential to mitigate the impacts of current (and future) epidemics; however, current methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require complicated laboratory tests that are generally conducted off-site and require substantial time. CRISPR-Cas systems have been harnessed to develop sensitive and specific platforms for nucleic acid detection. These detection platforms take advantage of CRISPR enzymes\u2019 RNA-guided specificity for RNA and DNA targets and collateral trans activities on single-stranded RNA and DNA reporters. Microbial genomes possess an extensive range of CRISPR enzymes with different specificities and levels of collateral activity; identifying new enzymes may improve CRISPR-based diagnostics. Here, we identified a new Cas13 variant, which we named as miniature Cas13 (mCas13), and characterized its catalytic activity. We then employed this system to design, build, and test a SARS-CoV-2 detection module coupling reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the mCas13 system to detect SARS-CoV-2 in synthetic and clinical samples. Our system exhibits sensitivity and specificity comparable to other CRISPR systems. This work expands the repertoire and application of Cas13 enzymes in diagnostics and for potential in vivo applications, including RNA knockdown and editing. Importantly, our system can be potentially adapted and used in large-scale testing for diverse pathogens, including RNA and DNA viruses, and bacteria.","lamp_id":[{"id":"LPB00393","pathogen":"SARS-CoV-2","target":"N"}]},{"id":208,"pmid":33354689,"title":"Rapid, point-of-care molecular diagnostics with Cas13","year":2021,"journal":"medRxiv","authors":"Shreeya Agrawal, Alison Fanton, Sita S. Chandrasekaran, B\u00e9r\u00e9nice Charrez, Arturo M. Escajeda, Sungmin Son, Roger Mcintosh, Abdul Bhuiya, Mar\u00eda D\u00edaz de Le\u00f3n Derby, Neil A. Switz, Maxim Armstrong, Andrew R. Harris, Noam Prywes, Maria Lukarska, Scott B. Biering, Dylan C. J. Smock, Amanda Mok, Gavin J. Knott, Qi Dang, Erik Van Dis, Eli Dugan, Shin Kim, Tina Y. Liu, IGI Testing Consortium, Eva Harris, Sarah A. Stanley, Liana F. Lareau, Ming X. Tan, Daniel A. Fletcher, Jennifer A. Doudna, David F. Savage, Patrick D. Hsu","doi":"10.1101\/2020.12.14.20247874","country":"","institute":"","deparment":"","abstract":"Rapid nucleic acid testing is a critical component of a robust infrastructure for increased disease surveillance. Here, we report a microfluidic platform for point-of-care, CRISPR-based molecular diagnostics. We first developed a nucleic acid test which pairs distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity. We combined this workflow with an extraction-free sample lysis protocol using shelf-stable reagents that are widely available at low cost, and a multiplexed human gene control for calling negative test results. As a proof-of-concept, we demonstrate sensitivity down to 40 copies\/\u03bcL of SARS-CoV-2 in unextracted saliva within 35 minutes, and validated the test on total RNA extracted from patient nasal swabs with a range of qPCR Ct values from 13\u201335. To enable sample-to-answer testing, we integrated this diagnostic reaction with a single-use, gravity-driven microfluidic cartridge followed by real-time fluorescent detection in a compact companion instrument. We envision this approach for Diagnostics with Coronavirus Enzymatic Reporting (DISCoVER) will incentivize frequent, fast, and easy testing.","lamp_id":[{"id":"LPB00394","pathogen":"SARS-CoV-2","target":"N"}]},{"id":209,"pmid":35027868,"title":"Development of a Saliva-Optimized RT-LAMP Assay for SARS-CoV-2","year":2021,"journal":"Journal of Biomolecular Techniques","authors":"Albert D. Yu, Kristina Galatsis, Jian Zheng, Jasmine Quynh Le, Dingbang Ma, Stanley Perlman, Michael Rosbash","doi":"10.7171\/jbt.21-3203-005","country":"USA","institute":"Brandeis University","deparment":"Howard Hughes Medical Institute and Department of Biology","abstract":"Conventional reverse transcription quantitative polymerase chain reaction (RT-qPCR) technology has struggled to fulfill the unprecedented need for diagnostic testing created by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Complexity and cost hinder access to testing, and long turnaround time decreases its utility. To ameliorate these issues, we focus on saliva and introduce several advances to colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology; RT-LAMP offers a minimal equipment alternative to RT-qPCR. First, we validated the use of the novel dye LAMPShade Violet (LSV), which improves the visual clarity and contrast of the colorimetric readout. Second, we compared different inactivation conditions on infectivity and RNA yield from saliva. Third, we developed a 10-minute RNA purification protocol from saliva. We call this magnetic bead protocol SalivaBeads. Finally, we developed a magnetic stick, StickLAMP, which provides reliable bead-based RNA purification as well as simple and low-cost access to scalable testing from saliva.","lamp_id":[{"id":"LPB00395","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00396","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":210,"pmid":34271398,"title":"A chemical-enhanced system for CRISPR-Based nucleic acid detection","year":2021,"journal":"Biosensors and Bioelectronics","authors":"Zihan Li, Wenchang Zhao, Shixin Ma, Zexu Li, Yingjia Yao, Teng Fei","doi":"10.1016\/j.bios.2021.113493","country":"People's Republic of China","institute":"Northeastern University\/Ministry of Education","deparment":"College of Life and Health Sciences\/Key Laboratory of Data Analytics and Optimization for Smart Industry (Northeastern University)","abstract":"The CRISPR-based nucleic acid detection systems have shown great potential for point-of-care testing of viral pathogens, especially in the context of COVID-19 pandemic. Here we optimize several key parameters of reaction chemistry and develop a Chemical Enhanced CRISPR Detection system for nucleic acid (termed CECRID). For the Cas12a\/Cas13a-based signal detection phase, we determine buffer conditions and substrate range for optimal detection performance, and reveal a crucial role of bovine serum albumin in enhancing trans-cleavage activity of Cas12a\/Cas13a effectors. By comparing several chemical additives, we find that addition of L-proline can secure or enhance Cas12a\/Cas13a detection capability. For isothermal amplification phase with typical LAMP and RPA methods, inclusion of L-proline can also enhance specific target amplification as determined by CRISPR detection. Using SARS-CoV-2 pseudovirus, we demonstrate CECRID has enhanced detection sensitivity over chemical additive-null method with either fluorescence or lateral flow strip readout. Thus, CECRID provides an improved detection power and system robustness, and helps to develop enhanced reagent formula or test kit towards practical application of CRISPR-based diagnostics.","lamp_id":[{"id":"LPB00397","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00398","pathogen":"SARS-CoV-2","target":"N"}]},{"id":211,"pmid":35433827,"title":"A Rapid, Highly Sensitive and Open-Access SARS-CoV-2 Detection Assay for Laboratory and Home Testing","year":2022,"journal":"Frontiers in Molecular Biosciences","authors":"Max J Kellner, James J Ross, Jakob Schnabl, Marcus P S Dekens, Martin Matl, Robert Heinen, Irina Grishkovskaya, Benedikt Bauer, Johannes Stadlmann, Luis Men\u00e9ndez-Arias, Andrew D Straw, Robert Fritsche-Polanz, Marianna Traugott, Tamara Seitz, Alexander Zoufaly, Manuela F\u00f6dinger, Christoph Wenisch, Johannes Zuber, Vienna COVID-19 Detection Initiative (VCDI), Andrea Pauli, Julius Brennecke","doi":"10.3389\/fmolb.2022.801309","country":"Austria\/UK","institute":"Vienna BioCenter (VBC)\/University of Vienna\/Cambridge","deparment":"Research Institute of Molecular Pathology (IMP)\/Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA)\/MRC Laboratory of Molecular Biology","abstract":"RT-qPCR-based diagnostic tests play important roles in combating virus-caused pandemics such as Covid-19. However, their dependence on sophisticated equipment and the associated costs often limits their widespread use. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative nucleic acid detection method that overcomes these limitations. Here, we present a rapid, robust, and sensitive RT-LAMP-based SARS-CoV-2 detection assay. Our 40-min procedure bypasses the RNA isolation step, is insensitive to carryover contamination, and uses a colorimetric readout that enables robust SARS-CoV-2 detection from various sample types. Based on this assay, we have increased sensitivity and scalability by adding a nucleic acid enrichment step (Bead-LAMP), developed a version for home testing (HomeDip-LAMP), and identified open-source RT-LAMP enzymes that can be produced in any molecular biology laboratory. On a dedicated website, rtlamp.org (DOI: 10.5281\/zenodo.6033689), we provide detailed protocols and videos. Our optimized, general-purpose RT-LAMP assay is an important step toward population-scale SARS-CoV-2 testing.","lamp_id":[{"id":"LPB00399","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00400","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00507","pathogen":"SARS-CoV-2","target":"E"}]},{"id":212,"pmid":33640656,"title":"Emulsion-based isothermal nucleic acid amplification for rapid SARS-CoV-2 detection via angle-dependent light scatter analysis","year":2021,"journal":"Biosensors and Bioelectronics","authors":"Alexander S. Day, Tiffany-Heather Ulep, Babak Safavinia, Tyler Hertenstein, Elizabeth Budiman, Laurel Dieckhaus, Jeong-Yeol Yoon","doi":"10.1016\/j.bios.2021.113099","country":"USA","institute":"The University of Arizona","deparment":"Department of Biomedical Engineering","abstract":"The SARS-CoV-2 pandemic, an ongoing global health crisis, has revealed the need for new technologies that integrate the sensitivity and specificity of RT-PCR tests with a faster time-to-detection. Here, an emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to allow for the compartmentalization of LAMP reactions, leading to faster changes in emulsion characteristics, and thus lowering time-to-detection. Within these droplets, ongoing LAMP reactions lead to adsorption of amplicons to the water-oil interface, causing a decrease in interfacial tension, resulting in smaller emulsion diameters. Changes in emulsion diameter allow for the monitoring of the reaction by use of angle-dependent light scatter (based off Mie scatter theory). Mie scatter simulations confirmed that light scatter intensity is diameter-dependent and smaller colloids have lower intensity values compared to larger colloids. Via spectrophotometers and fiber optic cables placed at 30\u00b0 and 60\u00b0, light scatter intensity was monitored. Scatter intensities collected at 5 min, 30\u00b0 could statistically differentiate 10, 10^3, and 10^5 copies\/\u03bcL initial concentrations compared to NTC. Similarly, 5 min scatter intensities collected at 60\u00b0 could statistically differentiate 105 copies\/\u03bcL initial concentrations in comparison to NTC. The use of both angles during the eLAMP assay allows for distinction between high and low initial target concentrations. The efficacy of a smartphone-based platform was also tested and had a similar limit of detection and assay time of less than 10 min. Furthermore, fluorescence-labeled primers were used to validate target nucleic acid amplification. Compared to existing LAMP assays for SARS-CoV-2 detection, these times-to-detections are very rapid.","lamp_id":[{"id":"LPB00401","pathogen":"SARS-CoV-2","target":"N"}]},{"id":213,"pmid":34109949,"title":"Can a field molecular diagnosis be accurate? A performance evaluation of colorimetric RT-LAMP for the detection of SARS-CoV-2 in a hospital setting","year":2021,"journal":"Analytical Methods","authors":"L\u00edvia do Carmo Silva, Carlos Abelardo Dos Santos, Geovana de Melo Mendes, K\u00e9zia Gomes de Oliveira, Marcio Neres de Souza J\u00fanior, Paulo Felipe Neves Estrela, S\u00e9rgio Henrique Nascente Costa, Elis\u00e2ngela de Paula Silveira-Lacerda, Gabriela Rodrigues Mendes Duarte","doi":"10.1039\/d1ay00481f","country":"Brazil","institute":"Universidade Federal de Goi\u00e1s","deparment":"Instituto de Qu\u00edmica","abstract":"SARS-CoV-2 currently represents a serious global public health problem. Non-pharmaceutical intervention measures (NPIs) have been widely adopted, and the testing strategy since the beginning of the infection is the most effective tool for tracking, isolating, and minimizing transmission. The high operating costs and the need for sophisticated instrumentation related to gold standard diagnostic for COVID-19, Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR), have highlighted the urgency and importance of developing and applying new diagnostic techniques, especially in places with scarce resources. Thus, alternative molecular tests, such as Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP), based on isothermal amplification have been used to detect SARS-CoV-2 using different protocols. The potential for field application of RT-LAMP is due to the lower cost and time and not requiring high-cost instrumentation. Here, we evaluate the colorimetric RT-LAMP to detect SARS-CoV-2 in a hospital environment and correlate its performance with tests performed in a reference laboratory. The analysis performed at the hospital showed high sensitivity (88.89%), specificity (98.55%), accuracy (95.83%), and a Cohen's kappa of 0.895. However, we achieved 100% of agreement when comparing the RT-LAMP results with the gold standard (qRT-PCR) results for samples with Ct < 30 in the hospital-based test. In addition, a similar performance was found in the field compared to the reference laboratory, corroborating the proposal to apply the test directly at point-of-care.","lamp_id":[{"id":"LPB00402","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"}]},{"id":214,"pmid":34044144,"title":"Evaluation of sample treatments in a safe and straightforward procedure for the detection of SARS-CoV-2 in saliva","year":2021,"journal":"International Journal of Infectious Diseases","authors":"Camila P Rubio, Lorena Franco-Mart\u00ednez, Cristina S\u00e1nchez Resalt, Alberto Torres-Cantero, Irene Martinez-Morata, Enrique Bernal, Mar\u00eda J Alcaraz, Mar\u00eda R Vicente-Romero, Silvia Mart\u00ednez-Subiela, Asta Tvarijonaviciute, Jos\u00e9 J Cer\u00f3n","doi":"10.1016\/j.ijid.2021.05.053","country":"Spain","institute":"University of Murcia","deparment":"Interdisciplinary Laboratory of Clinical Analysis","abstract":"Objectives: To evaluate four sample treatments in a safe and straightforward procedure to detect SARS-CoV-2 in saliva.\n\nMethods: Four sample treatments were evaluated in a 3-step procedure to detect SARS-CoV-2 in saliva: 1) heating at 95 \u00b0C for 5 min for sample inactivation; 2) sample treatment; 3) analysis by reverse-transcription loop-mediated isothermal amplification (LAMP). Saliva samples used were from infected individuals or were spiked with known quantities of viral particles.\n\nResults: Three treatments had a limit of detection (LOD) of 500.000 viral particles per ml of saliva and could be used to detect individuals with potential to transmit the disease. The treatment of phosphate buffer, dithiothreitol, ethylenediaminetetraacetic acid and proteinase K, with an additional 95 \u00b0C heating step, yielded a lower LOD of 95; its sensitivity ranged from 100% in patients with nasopharyngeal swab reverse-transcriptase polymerase chain reaction cycle threshold values <20 to 47.8% for values >30.\n\nConclusions: This report highlights the importance of an adequate sample treatment for saliva to detect SARS-CoV-2 and describes a flexible procedure that can be adapted to point-of-care. Although its sensitivity when LAMP is used is lower than reverse-transcriptase polymerase chain reaction, this procedure can contribute to COVID-19 control by detecting individuals able to transmit the disease.","lamp_id":[{"id":"LPB00403","pathogen":"SARS-CoV-2","target":"N"}]},{"id":215,"pmid":35860034,"title":"Modular micro-PCR system for the onsite rapid diagnosis of COVID-19","year":2022,"journal":"Microsystems & Nanoengineering","authors":"Phuong Quoc Mai Nguyen, Ming Wang, Nelisha Ann Maria, Adelicia Yongling Li, Hsih Yin Tan, Gordon Minru Xiong, Meng-Kwang Marcus Tan, Ali Asgar S Bhagat, Catherine W M Ong, Chwee Teck Lim","doi":"10.1038\/s41378-022-00400-3","country":"Singapore","institute":"National University of Singapore","deparment":"Institute for Health Innovation and Technology (iHealthtech)","abstract":"Effective containment of the COVID-19 pandemic requires rapid and accurate detection of the pathogen. Polymerase chain reaction (PCR) remains the gold standard for COVID-19 confirmation. In this article, we report the performance of a cost-effective modular microfluidic reverse transcription (RT)-PCR and RT-loop mediated isothermal amplification (RT-LAMP) platform, Epidax\u00ae, for the point-of-care testing and confirmation of SARS-CoV-2. This platform is versatile and can be reconfigured either for screening using endpoint RT-PCR or RT-LAMP tests or for confirmatory tests using real-time RT-PCR. Epidax\u00ae is highly sensitive and detects as little as 1 RNA copy per \u00b5L for real-time and endpoint RT-PCR, while using only half of the reagents. We achieved comparable results with those of a commercial platform when detecting SARS-CoV-2 viruses from 81 clinical RNA extracts. Epidax\u00ae can also detect SARS-CoV-2 from 44 nasopharyngeal samples without RNA extraction by using a direct RT-PCR assay, which shortens the sample-to-answer time to an hour with minimal user steps. Furthermore, we validated the technology using an RT-LAMP assay on 54 clinical RNA extracts. Overall, our platform provides a sensitive, cost-effective, and accurate diagnostic solution for low-resource settings.","lamp_id":[{"id":"LPB00404","pathogen":"SARS-CoV-2","target":"N"}]},{"id":216,"pmid":35856669,"title":"One-Pot Isothermal LAMP-CRISPR-Based Assay for Klebsiella pneumoniae Detection","year":2022,"journal":"Microbiology Spectrum","authors":"Xiaotong Qiu, Xueping Liu, Xiao Ma, Ruixue Wang, Shenglin Chen, Fang Li, Zhenjun Li","doi":"10.1128\/spectrum.01545-22","country":"China","institute":"National Institute for Communicable Disease Control and Prevention","deparment":"State Key Laboratory for Infectious Disease Prevention and Control","abstract":"Klebsiella pneumoniae (K. pneumoniae) is one of the most common pathogens causing nosocomial infection. A rapid, accurate, and convenient detection method is required for early diagnosis and directed therapy of K. pneumoniae infection. CRISPR-top (CRISPR-mediated testing in one pot) is a LAMP-CRISPR-based nucleic acid detection platform, which integrates target preamplification with CRISPR\/Cas12b-based detection into a one-pot reaction mixture, performed at a constant temperature. In this study, we established the K. pneumoniae CRISPR-top assay to precisely identify K. pneumoniae at 56\u00b0C within 60 min. The reaction mixture with 0.53 \u03bcM (each) FIP and BIP, 0.27 \u03bcM LF, 0.13 \u03bcM (each) F3 and B3, and 2 \u03bcM ssDNA fluorescence probe was determined as the optimal reaction system of our assay. The limit of detection of this assay is 1 pg genomic DNA (equivalent to 160 K. pneumoniae cells and 1.6 \u00d7 10^5 CFU\/mL for samples) per reaction, which is 10-fold more sensitive than LAMP. Up to 105 strains composed of K. pneumoniae clinical isolates and non-K. pneumoniae strains were correctly identified by our assay. A total of 58 sputum samples collected from patients with respiratory symptoms were used to evaluate the diagnostic performance of the K. pneumoniae CRISPR-top assay. As a result, the K. pneumoniae CRISPR-top assay yielded 100% (33\/33) specificity and 96% (24\/25) sensitivity, as well as a positive predictive value of 100% (24\/24) and a negative predictive value of 97.1% (33\/34), which were all higher than LAMP detection. In conclusion, the K. pneumoniae CRISPR-top assay developed in this study is a simple, rapid and ultra-specific method to detect K. pneumoniae. ","lamp_id":[{"id":"LPB00405","pathogen":"Klebsiella pneumoniae subsp. pneumoniae NTUH-K2044","target":"rcsA"}]},{"id":217,"pmid":34903068,"title":"High-quality RNA improves sensitivity of SARS-CoV-2 detection by colorimetric RT-LAMP","year":2021,"journal":"Experimental Biology and Medicine","authors":"Marta Puigmul\u00e9, M\u00f2nica Coll, Alexandra P\u00e9rez-Serra, Laura L\u00f3pez, Ferran Pic\u00f3, Nuria Neto, M\u00f2nica Corona, Mel\u00b7lina Pinsach-Abuin, Carles Ferrer-Costa, Maria Bux\u00f3, Francesc-Xavier Queralt, Ramon Brugada","doi":"10.1177\/15353702211054768","country":"","institute":"","deparment":"","abstract":"The global SARS-CoV-2 pandemic requires a rapid, reliable, and user-friendly diagnostic test to help control the spread of the virus. Reverse transcription and quantitative PCR (RT-qPCR) is currently the gold standard method for SARS-CoV-2 detection. Here, we develop a protocol based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and demonstrate increased sensitivity of this technique using fresh RNA extracts compared to RNA samples subjected to freezing\/thawing cycles. We further compare RT-LAMP to RT-qPCR and demonstrate that the RT-LAMP approach has high sensitivity in fresh RNA extracts and can detect positive samples with Ct values between 8 and 35.","lamp_id":[{"id":"LPB00406","pathogen":"SARS-CoV-2","target":"N"}]},{"id":218,"pmid":30881058,"title":"Loop-mediated isothermal amplification for detection of Legionella pneumophila in respiratory specimens of hospitalized patients in Ahvaz, southwest Iran","year":2019,"journal":"Infection and Drug Resistance","authors":"Mojtaba Moosavian, Sakineh Seyed-Mohammadi, Morteza Saki, Fatemeh Shahi, Mahtab Khoshkholgh Sima, Davoud Afshar, Sara Barati","doi":"10.2147\/IDR.S198099","country":"","institute":"","deparment":"","abstract":"Background: Legionnaires' disease is an important public health problem that can cause substantial mortality and morbidity. Legionnaires' disease-risk estimation may be compromised by uncertainties in Legionella-detection methods. The aim of this study was the detection of L. pneumophila in respiratory specimens of hospitalized patients with respiratory symptoms by culture, PCR, and loop-mediated isothermal amplification (LAMP) methods.\n\nMethods: Sputum and bronchoalveolar lavage samples were obtained from patients with pneumonia admitted to teaching hospitals in Ahvaz, Iran from June 2016 to March 2017. Isolation of Legionella spp. was done by culturing the samples directly onto buffered charcoal-yeast extract and modified Wadowsky-Yee agar medium. Then, PCR and LAMP assays were performed for detection of L. pneumophila via its mip gene in respiratory specimens.\n\nResults: A total of 100 respiratory specimens were collected. Our results showed that 1% of the samples were culture positive for Legionella spp., and 3% and 7% of samples were positive for L. pneumophila using the mip gene on PCR and LAMP assays, respectively.\n\nConclusion: Legionnaires' disease should be considered in the diagnosis of pulmonary infectious diseases. Also, the LAMP assay is a faster method with higher sensitivity and specificity than conventional methods, such as PCR and culture, for laboratory diagnosis of Legionnaires' disease.","lamp_id":[{"id":"LPB00407","pathogen":"Legionella pneumophila","target":"mip"}]},{"id":219,"pmid":null,"title":"Analysis, quantification, and visualization of RT-LAMP technique for detection of COVID-19","year":2021,"journal":"medRxiv","authors":"Mohammad H Ghazimoradi, Maryam Daryani, Masoud Garshasbi, Ehsan Zolghadr, Ali Khalafizadeh, Sadegh Babashah","doi":"10.1101\/2021.07.15.21260528","country":"Iran","institute":"Tarbiat Modares University","deparment":"Department of Molecular Genetics","abstract":"Background SARS-Cov-2 is a new virus that caused an epidemic disease, COVID-19. According to the world health organization, detecting the patients\/carriers is by the far the most important action to prevent the pandemic. Recently, the loop-mediated isothermal amplification (LAMP) technique has become more popular due to the easy handling of a one-step kit used for the detection of many diseases than RT-PCR-based techniques.\n\nmethods Herein, we used the RT-LAMP technique so as to detect COVID-19. To this end, 40 paired-samples of patients and healthy people had been collected and tested by RT-PCR for N and E genes of SARS-CoV-2. The RT-LAMP test has been performed on samples for the RdRp gene. The sensitivity and specificity of tests have been determined.\n\nResults The testing results are consistent with the conventional RT-qPCR. Additionally, we also showed that a one-step process without RNA extraction is feasible to achieve RNA amplification directly from a sample.\n\nConclusion We confirmed that RT-LAMP is a rapid, simple, and sensitive method that can be used as a large-screening method, particularly in regional hospitals with limited access to high-technologies.","lamp_id":[{"id":"LPB00408","pathogen":"SARS-CoV-2","target":"RdRp"}]},{"id":220,"pmid":33782112,"title":"Diagnosis of SARS-CoV-2 Infection with LamPORE, a High-Throughput Platform Combining Loop-Mediated Isothermal Amplification and Nanopore Sequencing","year":2021,"journal":"Journal of Clinical Microbiology","authors":"Leon Peto, Gillian Rodger, Daniel P Carter, Karen L Osman, Mehmet Yavuz, Katie Johnson, Mohammad Raza, Matthew D Parker, Matthew D Wyles, Monique Andersson, Anita Justice, Alison Vaughan, Sarah Hoosdally, Nicole Stoesser, Philippa C Matthews, David W Eyre, Timothy E A Peto, Miles W Carroll, Thushan I de Silva, Derrick W Crook, Cariad M Evans, Steven T Pullan","doi":"10.1128\/JCM.03271-20","country":"United Kingdom","institute":"Oxford University Hospitals NHS Foundation Trust\/University of Oxford\/National Infection Service","deparment":"Nuffield Department of Medicine\/Public Health England","abstract":"LamPORE is a novel diagnostic platform for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA combining loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyze thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against reverse transcriptase PCR (RT-PCR) using RNA extracted from spiked respiratory samples and stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 10 genome copies\/\u03bcl of extracted RNA, which is above the limit achievable by RT-PCR, but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among them, LamPORE had a diagnostic sensitivity of 99.1% (226\/228; 95% confidence interval [CI], 96.9% to 99.9%). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278\/279; 98.0% to 100.0%). Overall, 1.4% (7\/514; 0.5% to 2.9%) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478\/494; 94.8% to 98.1%). LamPORE has a similar performance as RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients and offers a promising approach to high-throughput testing.","lamp_id":[{"id":"LPB00410","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00411","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00412","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":221,"pmid":34183720,"title":"A rapid near-patient detection system for SARS-CoV-2 using saliva","year":2021,"journal":"Scientific Reports","authors":"Noah B. Toppings, Abu Naser Mohon, Yoonjung Lee, Hitendra Kumar, Daniel Lee, Ratik Kapoor, Gurmukh Singh, Lisa Oberding, Omar Abdullah, Keekyoung Kim, Byron M. Berenger, Dylan R. Pillai","doi":"10.1038\/s41598-021-92677-z","country":"Canada","institute":"University of Calgary","deparment":"Department of Microbiology, Immunology, and Infectious Diseases","abstract":"The highly infectious nature of SARS-CoV-2 necessitates the use of widespread testing to control the spread of the virus. Presently, the standard molecular testing method (reverse transcriptase-polymerase chain reaction, RT-PCR) is restricted to the laboratory, time-consuming, and costly. This increases the turnaround time for getting test results. This study sought to develop a rapid, near-patient saliva-based test for COVID-19 (Saliva-Dry LAMP) with similar accuracy to that of standard RT-PCR tests. A lyophilized dual-target reverse transcription-loop-mediated isothermal amplification (RT-LAMP) test with fluorometric detection by the naked eye was developed. The assay relies on dry reagents that are room temperature stable. A device containing a centrifuge, heat block, and blue LED light system was manufactured to reduce the cost of performing the assay. This test has a limit of detection of 1 copy\/\u00b5L and achieved a positive percent agreement of 100% [95% CI 88.43% to 100.0%] and a negative percent agreement of 96.7% [95% CI 82.78-99.92%] relative to a reference standard test. Saliva-Dry LAMP can be completed in 105 min. Precision, cross-reactivity, and interfering substances analysis met international regulatory standards. The combination of ease of sample collection, dry reagents, visual detection, low capital equipment cost, and excellent analytical sensitivity make Saliva-Dry LAMP particularly useful for resource-limited settings.","lamp_id":[{"id":"LPB00413","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00414","pathogen":"SARS-CoV-2","target":"RdRp"}]},{"id":222,"pmid":34033472,"title":"Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Ultrasensitive Detection of SARS-CoV-2 in Saliva and Viral Transport Medium Clinical Samples","year":2021,"journal":"Analytical Chemistry","authors":"Anurup Ganguli, Ariana Mostafa, Jacob Berger, Jongwon Lim, Elbashir Araud, Janice Baek, Sarah A Stewart de Ramirez, Ali Baltaji, Kelly Roth, Muhammad Aamir, Surya Aedma, Mohamed Mady, Pranav Mahajan, Sanjivani Sathe, Mark Johnson, Karen White, James Kumar, Enrique Valera, Rashid Bashir","doi":"10.1021\/acs.analchem.0c05170","country":"USA","institute":"University of Illinois at Urbana-Champaign","deparment":"Department of Bioengineering\/Nick Holonyak Jr. Micro and Nanotechnology Laboratory","abstract":"The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostics platforms. Although several polymerase chain reaction (PCR)-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. In addition, there are limited reports of saliva as the sample source, and some of these indicate inferior sensitivity when comparing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay from saliva using a two-step RT-LAMP assay, where a short 10 min RT step is performed with only B3 and backward inner primers before the final reaction. We show that while the one-step RT-LAMP demonstrates satisfactory results, the optimized two-step approach allows detection of only few molecules per reaction and performs significantly better than the one-step RT-LAMP and conventional two-step RT-LAMP approaches with all primers included in the RT step. We show control measurements with RT-PCR, and importantly, we demonstrate RNA extraction-free RT-LAMP-based assays for detection of SARS-CoV-2 from viral transport media and saliva clinical samples.","lamp_id":[{"id":"LPB00415","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00416","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00417","pathogen":"SARS-CoV-2","target":"ORF8"},{"id":"LPB00418","pathogen":"SARS-CoV-2","target":"N"}]},{"id":223,"pmid":34922321,"title":"Developing RT-LAMP assays for rapid diagnosis of SARS-CoV-2 in saliva","year":2022,"journal":"EBioMedicine","authors":"Xin Huang, Gongyu Tang, Nahed Ismail, Xiaowei Wang","doi":"10.1016\/j.ebiom.2021.103736","country":"USA","institute":"University of Illinois Cancer Center","deparment":"Department of Pharmacology and Regenerative Medicine","abstract":"Background: The coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has killed millions of people worldwide. The current crisis has created an unprecedented demand for rapid test of SARS-CoV-2 infection.\n\nMethods: Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a fast and convenient method to amplify and identify the transcripts of a targeted pathogen. However, the sensitivity and specificity of RT-LAMP were generally regarded as inferior when compared with the gold standard RT-qPCR. To address this issue, we combined bioinformatic and experimental analyses to improve the assay performance for COVID-19 diagnosis.\n\nFindings: First, by experimental screening as well as high-throughput sequencing studies, we discovered new primer features that impacted LAMP sensitivity and specificity. These features were then used to build an improved bioinformatics algorithm to design LAMP primers targeting SARS-CoV-2. We further rigorously validated these new assays for their efficacy and specificity. We demonstrated that multiplexed RT-LAMP assay could directly detect as low as 1.5 copies\/\u00b5L of SARS-CoV-2 particles in saliva, without the need of RNA isolation. We further tested this ultra-sensitive and specific RT-LAMP assay using saliva samples from COVID-19 patients. Clinical validation results indicated that the new RT-LAMP assay was comparable to standard RT-qPCR in overall assay sensitivity and specificity.\n\nInterpretation: In summary, our new LAMP primer design algorithm along with the validated assays provide a fast and reliable method for the diagnosis of COVID-19 cases.","lamp_id":[{"id":"LPB00419","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00420","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00421","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00422","pathogen":"SARS-CoV-2","target":"M"},{"id":"LPB00423","pathogen":"SARS-CoV-2","target":"M"}]},{"id":224,"pmid":34385527,"title":"A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples","year":2021,"journal":"Scientific Reports","authors":"Catarina Amaral, Wilson Antunes, Elin Moe, Am\u00e9rico G Duarte, Lu\u00eds M P Lima, Cristiana Santos, In\u00eas L Gomes, Gon\u00e7alo S Afonso, Ricardo Vieira, Helena Sofia S Teles, Marisa S Reis, Manuel A Ramalho da Silva, Ana Margarida Henriques, Miguel Fevereiro, M Rita Ventura, M\u00f3nica Serrano, Catarina Pimentel","doi":"10.1038\/s41598-021-95799-6","country":"Portugal","institute":"Universidade Nova de Lisboa","deparment":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier","abstract":"Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.","lamp_id":[{"id":"LPB00424","pathogen":"SARS-CoV-2","target":"N"}]},{"id":225,"pmid":34423284,"title":"A paper-based colorimetric molecular test for SARS-CoV-2 in saliva","year":2021,"journal":"Biosensors and Bioelectronics: X","authors":"Josiah Levi Davidson, Jiangshan Wang, Murali Kannan Maruthamuthu, Andres Dextre, Ana Pascual-Garrigos, Suraj Mohan, Sai Venkata Sravan Putikam, Fujr Osman Ibrahim Osman, Darby McChesney, Jordan Seville, Mohit S Verma","doi":"10.1016\/j.biosx.2021.100076","country":"USA","institute":"Purdue University","deparment":"Department of Agricultural and Biological Engineering\/Birck Nanotechnology Center","abstract":"Herein, we describe the development of a paper-based device to detect nucleic acids of pathogens of interest in complex samples using loop-mediated isothermal amplification (LAMP) by producing a colorimetric response visible to the human eye. To demonstrate the utility of this device in emerging public health emergencies, we developed and optimized our device to detect SARS-CoV-2 in human saliva without preprocessing. The resulting device was capable of detecting the virus within 60 min and had an analytical sensitivity of 97% and a specificity of 100% with a limit of detection of 200 genomic copies\/\u03bcL of patient saliva using image analysis. The device consists of a configurable number of reaction zones constructed of Grade 222 chromatography paper separated by 20 mil polystyrene spacers attached to a Melinex\u00ae backing via an ARclean\u00ae double-sided adhesive. The resulting device is easily configurable to detect multiple targets and has the potential to detect a variety of pathogens simply by changing the LAMP primer sets.","lamp_id":[{"id":"LPB00425","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00426","pathogen":"SARS-CoV-2","target":"ORF7a"}]},{"id":226,"pmid":34910507,"title":"Harmony COVID-19: A ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection","year":2021,"journal":"Science Advances","authors":"Nuttada Panpradist, Enos C Kline, Robert G Atkinson, Michael Roller, Qin Wang, Ian T Hull, Jack H Kotnik, Amy K Oreskovic, Crissa Bennett, Daniel Leon, Victoria Lyon, Shane D Gilligan-Steinberg, Peter D Han, Paul K Drain, Lea M Starita, Matthew J Thompson, Barry R Lutz","doi":"10.1126\/sciadv.abj1281","country":"USA","institute":"University of Washington","deparment":"Department of Bioengineering\/Global Health for Women, Adolescents, and Children","abstract":"RNA amplification tests sensitively detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but their complexity and cost are prohibitive for expanding coronavirus disease 2019 (COVID-19) testing. We developed \u201cHarmony COVID-19,\u201d a point-of-care test using inexpensive consumables, ready-to-use reagents, and a simple device. Our ready-to-use, multiplexed reverse transcription, loop-mediated isothermal amplification (RT-LAMP) can detect down to 0.38 SARS-CoV-2 RNA copies\/\u03bcl and can report in 17 min for high\u2013viral load samples (5000 copies\/\u03bcl). Harmony detected 97 or 83% of contrived samples with \u22650.5 viral particles\/\u03bcl in nasal matrix or saliva, respectively. Evaluation in clinical nasal specimens (n = 101) showed 100% detection of RNA extracted from specimens with \u22650.5 SARS-CoV-2 RNA copies\/\u03bcl, with 100% specificity in specimens positive for other respiratory pathogens. Extraction-free analysis (n = 29) had 95% success in specimens with \u22651 RNA copies\/\u03bcl. Usability testing performed first time by health care workers showed 95% accuracy.","lamp_id":[{"id":"LPB00427","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00428","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00429","pathogen":"SARS-CoV-2","target":"N"}]},{"id":227,"pmid":35708340,"title":"Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes","year":2022,"journal":"Microbiology Spectrum","authors":"Enos C Kline, Nuttada Panpradist, Ian T Hull, Qin Wang, Amy K Oreskovic, Peter D Han, Lea M Starita, Barry R Lutz","doi":"10.1128\/spectrum.01583-21","country":"USA","institute":"University of Washingtongrid","deparment":"Department of Bioengineering","abstract":"The increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high-throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false-negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multitarget redundancy and an internal amplification control. A convenient and cost-effective target-specific fluorescence detection system allows amplifications to be grouped by signal using adaptable probes for pooled reporting of SARS-CoV-2 target amplifications or differentiation of the Internal Amplification Control. Over the course of the pandemic, primer coverage of viral lineages by the three redundant sub-assays has varied from assay to assay as they have diverged from the Wuhan-Hu-1 isolate sequence, but aggregate coverage has remained high for all variant sequences analyzed, with a minimum of 97.4% (Variant of Interest: Eta). In three instances (Delta, Gamma, Eta), a high-frequency mismatch with one of the three sub-assays was observed, but overall coverage remained high due to multitarget redundancy. When challenged with extracted human samples the multiplex assay showed 87% or better sensitivity (of 30 positive samples), with 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction (of 21 positive samples), and 100% specificity (of 60 negative samples). These results are further evidence that conventional laboratory methodologies can be leveraged at the point of care for robust performance and diagnostic stability over time.","lamp_id":[{"id":"LPB00430","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00431","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00432","pathogen":"SARS-CoV-2","target":"N"}]},{"id":228,"pmid":31620745,"title":"A sample-to-answer, portable platform for rapid detection of pathogens with a smartphone interface","year":2019,"journal":"Lab on a Chip","authors":"Yu-Dong Ma, Kuang-Hsien Li, Yi-Hong Chen, Yung-Mao Lee, Shang-Ta Chou, Yue-Yuan Lai, Po-Chiun Huang, Hsi-Pin Ma, Gwo-Bin Lee","doi":"10.1039\/c9lc00797k","country":"Taiwan","institute":"National Tsing Hua University","deparment":"Department of Power Mechanical Engineering","abstract":"Emerging and re-emerging infectious diseases pose global threats to human health. Although several conventional diagnostic methods have been widely adopted in the clinic, the long turn-around times of \"gold standard\" culture-based techniques, as well as the limited sensitivity of lateral-flow strip assays, thwart medical progress. In this study, a smartphone-controlled, automated, and portable system was developed for rapid molecular diagnosis of pathogens (including viruses and bacteria) via the use of a colorimetric loop-mediated isothermal amplification (LAMP) approach on a passive, self-driven microfluidic device. The system was capable of 1) purifying viral or bacterial samples with specific affinity reagents that had been pre-conjugated to magnetic beads, 2) lysing pathogens at low temperatures, 3) executing isothermal nucleic acid amplification, and 4) quantifying the results of colorimetric assays for detection of pathogens with an integrated color sensor. The entire, 40 min analytical process was automatically performed with a novel punching-press mechanism that could be controlled and monitored by a smartphone. As a proof of concept, the influenza A (H1N1) virus and methicillin-resistant Staphylococcus aureus bacteria were used to characterize and optimize the device, and the limits of detection were experimentally found to be 3.2 \u00d7 10^-3 hemagglutinating units (HAU) per reaction and 30 colony-forming units (CFU) per reaction, respectively; both such values represent high enough sensitivity for clinical adoption. Moreover, the colorimetric assay could be both qualitative and quantitative for detection of pathogens. This is the first instance of an easy-to-use, automated, and portable system for accurate and sensitive molecular diagnosis of either viruses or bacteria, and it is envisioned that this smartphone-controlled apparatus may serve as a platform for clinical, point-of-care pathogen detection, particularly in resource-limited settings.","lamp_id":[{"id":"LPB00433","pathogen":"Influenza A virus (H1N1)","target":"HA"}]},{"id":229,"pmid":34482896,"title":"A lab-on-a-chip platform for integrated extraction and detection of SARS-CoV-2 RNA in resource-limited settings","year":2021,"journal":"Analytica Chimica Acta","authors":"Pablo Rodriguez-Mateos, Bongkot Ngamsom, Cheryl Walter, Charlotte E Dyer, Jesse Gitaka, Alexander Iles, Nicole Pamme","doi":"10.1016\/j.aca.2021.338758","country":"United Kingdom","institute":"University of Hull","deparment":"Department of Chemistry and Biochemistry","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the unprecedented global pandemic of coronavirus disease-2019 (COVID-19). Efforts are needed to develop rapid and accurate diagnostic tools for extensive testing, allowing for effective containment of the infection via timely identification and isolation of SARS-CoV-2 carriers. Current gold standard nucleic acid tests require many separate steps that need trained personnel to operate specialist instrumentation in laboratory environments, hampering turnaround time and test accessibility, especially in low-resource settings. We devised an integrated on-chip platform coupling RNA extraction based on immiscible filtration assisted by surface tension (IFAST), with RNA amplification and detection via colorimetric reverse-transcription loop mediated isothermal amplification (RT-LAMP), using two sets of primers targeting open reading frame 1a (ORF1a) and nucleoprotein (N) genes of SARS-CoV-2. Results were identified visually, with a colour change from pink to yellow indicating positive amplification, and further confirmed by DNA gel electrophoresis. The specificity of the assay was tested against HCoV-OC43 and H1N1 RNAs. The assay based on use of gene N primers was 100% specific to SARS-CoV-2 with no cross-reactivity to HCoV-OC43 nor H1N1. Proof-of-concept studies on water and artificial sputum containing genomic SARS-CoV-2 RNA showed our IFAST RT-LAMP device to be capable of extracting and detecting 470 SARS-CoV-2 copies mL-1 within 1 h (from sample-in to answer-out). IFAST RT-LAMP is a simple-to-use, integrated, rapid and accurate COVID-19 diagnostic platform, which could provide an attractive means for extensive screening of SARS-CoV-2 infections at point-of-care, especially in resource-constrained settings.","lamp_id":[{"id":"LPB00434","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00435","pathogen":"SARS-CoV-2","target":"N"}]},{"id":230,"pmid":34867841,"title":"Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern","year":2021,"journal":"Frontiers in Microbiology","authors":"Pedro A. Alves, de Ellen G. Oliveira, Ana Paula M. Franco-Luiz, Let\u00edcia T. Almeida, Amanda B. Gon\u00e7alves, Iara A. Borges, Fl\u00e1via de S. Rocha, Raissa P. Rocha, Matheus F. Bezerra, P\u00e2mella Miranda, Fl\u00e1vio D. Capanema, Henrique R. Martins, Gerald Weber, Santuza M. R. Teixeira, Gabriel Luz Wallau, Rubens L. do Monte-Neto","doi":"10.3389\/fmicb.2021.713713","country":"Brazil","institute":"Instituto Ren\u00e9 Rachou \u2013 Funda\u00e7\u00e3o Oswaldo Cruz\/Centro de Tecnologia em Vacinas","deparment":"","abstract":"The coronavirus disease 2019 (COVID-19) pandemic unfolded due to the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 \u00b1 2.7 viral genomic copies\/\u03bcL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65\u00b0C for 30 min. When compared to reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), up to cycle-threshold (Ct) value 32, RT-LAMP presented 98% [95% confidence interval (CI) = 95.3-99.5%] sensitivity and 100% (95% CI = 94.5-100%) specificity for SARS-CoV-2 RNA detection targeting E and N genes. No cross-reactivity was detected when testing other non-SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction-free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern and variants of interest, such as variants occurring in Brazil named gamma (P.1), zeta (P.2), delta (B.1.617.2), B.1.1.374, and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipment, infrastructure, and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive, and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.","lamp_id":[{"id":"LPB00436","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00437","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00438","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00439","pathogen":"SARS-CoV-2","target":"E"}]},{"id":231,"pmid":null,"title":"Clinical validation of RCSMS: a rapid and sensitive CRISPR-Cas12a test for the molecular detection of SARS-CoV-2 from saliva","year":2021,"journal":"medRxiv","authors":"Joaqu\u00edn Abugatt\u00e1s N\u00fa\u00f1ez del Prado, Ang\u00e9lica Quintana Reyes, Juan Blume La Torre, Renzo Guti\u00e9rrez Loli, Alejandro Pinz\u00f3n Olejua, Elena Roc\u00edo Chamorro Chirinos, F\u00e9lix Antonio Loza Mauricio, Jorge L. Magui\u00f1a, Julio Leon, Piere Rodr\u00edguez Aliaga, Edward M\u00e1laga Trillo","doi":"10.1101\/2021.04.26.21256081","country":"Peru","institute":"Universidad Peruana Cayetano Heredia","deparment":"Facultad de Ciencias y Filosof\u00eda","abstract":"Early detection of SARS-CoV-2 using molecular techniques is paramount to the fight against COVID-19. Due to its high sensitivity and specificity, RT-qPCR is the \u201cgold standard\u201d method for this purpose. However, its technical requirements, processing time and elevated costs hamper its use towards massive and timely molecular testing for COVID-19 in rural and socioeconomically deprived areas of Latin America. The advent and rapid evolution of CRISPR-Cas technology has boosted the development of new pathogen detection methodologies. Recently, DETECTR -a combination of isothermal RT-LAMP amplification and Cas12a-mediated enzymatic detection-has been successfully validated in the Netherlands and the USA as a rapid and low-cost alternative to RT-qPCR for the detection of SARS-CoV-2 from nasopharyngeal swabs. Here, we evaluated the performance of RCSMS, a locally adapted variant of DETECTR, to ascertain the presence of SARS-CoV-2 in saliva samples from 276 patients in two hospitals in Lima, Per\u00fa (current status over a total of 350 samples). We show that a low-cost thermochemical treatment with TCEP\/EDTA is sufficient to inactivate viral particles and cellular nucleases in saliva, eliminating the need to extract viral RNA with commercial kits, as well as the cumbersome nasopharyngeal swab procedure and the requirement of biosafety level 2 laboratories for molecular analyses. Our clinical validation shows that RCSMS detects up to 5 viral copies per reaction in 40 min, with sensitivity and specificity of 93.8% and 99.0% in the field, respectively, relative to RT-qPCR. Since CRISPR-Cas biosensors can be easily reprogrammed by using different guide RNA molecules, RCSMS has the potential to be quickly adapted for the detection of new SARS-CoV-2 variants. Notably, estimation of its negative and positive predictive values suggests that RCSMS can be confidently deployed in both high and low prevalence settings. Furthermore, our field study validates the use of lateral flow strips to easily visualize the presence of SARS-CoV-2, which paves the way to deploy RCSMS as a \u201cpoint of care\u201d test in environments with limited access to state-of-the-art diagnostic laboratories. In sum, RCSMS is a fast, efficient and inexpensive alternative to RT-qPCR for expanding COVID-19 testing capacity in low- and middle-income countries.","lamp_id":[{"id":"LPB00440","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00441","pathogen":"SARS-CoV-2","target":"E"}]},{"id":232,"pmid":34677329,"title":"Rapid COVID-19 Molecular Diagnostic System Using Virus Enrichment Platform","year":2021,"journal":"Biosensors","authors":"Yoon Ok Jang, Hyo Joo Lee, Bonhan Koo, Hye\u2010Hee Cha, Ji\u2010Soo Kwon, Ji Yeun Kim, Myoung Gyu Kim, Hyun Soo Kim, Sung\u2010Han Kim, Yong Shin","doi":"10.3390\/bios11100373","country":"Korea","institute":"Yonsei University","deparment":"Department of Biotechnology","abstract":"The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus (SARS-CoV)-2, is rapidly spreading and severely straining the capacities of public health communities and systems around the world. Therefore, accurate, rapid, and robust diagnostic tests for COVID-19 are crucial to prevent further spread of the infection, alleviate the burden on healthcare and diagnostic facilities, and ensure timely therapeutic intervention. To date, several detection methods based on nucleic acid amplification have been developed for the rapid and accurate detection of SARS-CoV-2. Despite the myriad of advancements in the detection methods for SARS-CoV-2, rapid sample preparation methods for RNA extraction from viruses have rarely been explored. Here, we report a rapid COVID-19 molecular diagnostic system that combines a self-powered sample preparation assay and loop-mediated isothermal amplification (LAMP) based naked-eye detection method for the rapid and sensitive detection of SARS-CoV-2. The self-powered sample preparation assay with a hydrophilic polyvinylidene fluoride filter and dimethyl pimelimidate can be operated by hand, without the use of any sophisticated instrumentation, similar to the reverse transcription (RT)-LAMP-based lateral flow assay for the naked-eye detection of SARS-CoV-2. The COVID-19 molecular diagnostic system enriches the virus population, extracts and amplifies the target RNA, and detects SARS-CoV-2 within 60 min. We validated the accuracy of the system by using 23 clinical nasopharyngeal specimens. We envision that this proposed system will enable simple, facile, efficient, and inexpensive diagnosis of COVID-19 at home and the clinic as a pre-screening platform to reduce the burden on the medical staff in this pandemic era.","lamp_id":[{"id":"LPB00442","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00443","pathogen":"SARS-CoV-2","target":"N"}]},{"id":233,"pmid":34534259,"title":"Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs","year":2021,"journal":"PLoS One","authors":"Les Jones, Abhijeet Bakre, Hemant Naikare, Ravindra Kolhe, Susan Sanchez, Yung-Yi C Mosley, Ralph A Tripp","doi":"10.1371\/journal.pone.0257563","country":"USA","institute":"University of Georgia\/State of Georgia COVID-19 Taskforce","deparment":"Department of Infectious Disease","abstract":"The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41\/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59\/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RT-LAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1\/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity.","lamp_id":[{"id":"LPB00444","pathogen":"SARS-CoV-2","target":"N"}]},{"id":234,"pmid":34541844,"title":"Single- and Two-Stage, Closed-Tube, Point-of-Care, Molecular Detection of SARS-CoV-2","year":2021,"journal":"Analytical Chemistry","authors":"Jinzhao Song, Mohamed El-Tholoth, Yize Li, Jevon Graham-Wooten, Yining Liang, Juan Li, Weijian Li, Susan R Weiss, Ronald G Collman, Haim H Bau","doi":"10.1021\/acs.analchem.1c03016","country":"China\/USA","institute":"Institute of Basic Medicine and Cancer (IBMC)\/University of Pennsylvania","deparment":"The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital)\/Department of Mechanical Engineering and Applied Mechanics","abstract":"Short of a vaccine, frequent and rapid testing, preferably at home, is the most effective strategy to contain the COVID-19 pandemic. Herein, we report on single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation. Our single-stage amplification is reverse transcription-loop mediated isothermal amplification (RT-LAMP) with custom-designed primers targeting the ORF1ab and the N gene regions of the virus genome. Our new two-stage amplification, dubbed Penn-RAMP, comprises recombinase isothermal amplification (RT-RPA) as its first stage and LAMP as its second stage. We compared various sample preparation strategies aimed at deactivating the virus while preserving its RNA and tested contrived and patient samples, consisting of nasopharyngeal swabs, oropharyngeal swabs, and saliva. Amplicons were detected either in real time with fluorescent intercalating dye or after amplification with the intercalating colorimetric dye LCV, which is insensitive to sample's PH. Our single RT-LAMP tests can be carried out instrumentation-free. To enable concurrent testing of multiple samples, we developed an inexpensive heat block that supports both the single-stage and two-stage amplification. Our RT-LAMP and Penn-RAMP assays have, respectively, analytical sensitivities of 50 and 5 virions\/reaction. Both our single- and two-stage assays have successfully detected SARS-CoV-2 in patients with viral loads corresponding to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) threshold cycle smaller than 32 while operating with minimally processed samples, without nucleic acid isolation. Penn-RAMP provides a 10-fold better sensitivity than RT-LAMP and does not need thermal cycling like PCR assays. All reagents are amenable to dry, refrigeration-free storage. The SARS-CoV-2 test described herein is suitable for screening at home, at the point of need, and in resource-poor settings.","lamp_id":[{"id":"LPB00445","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00446","pathogen":"SARS-CoV-2","target":"N"}]},{"id":235,"pmid":34571479,"title":"An internet of things-based point-of-care device for direct reverse-transcription-loop mediated isothermal amplification to identify SARS-CoV-2","year":2022,"journal":"Biosensors and Bioelectronics","authors":"Huynh Quoc Nguyen, Hoang Khang Bui, Vu Minh Phan, Tae Seok Seo","doi":"10.1016\/j.bios.2021.113655","country":"South Korea","institute":"Kyung Hee University","deparment":"Department of Chemical Engineering (BK21 FOUR Integrated Engineering Program)","abstract":"Rapid and accurate testing tools for SARS-CoV-2 detection are urgently needed to prevent the spreading of the virus and to take timely governmental actions. Internet of things (IoT)-based diagnostic devices would be an ideal platform for point-of-care (POC) screening of COVID-19 and ubiquitous healthcare monitoring for patients. Herein, we present an advanced IoT-based POC device for real-time direct reverse-transcription-loop mediated isothermal amplification assay to detect SARS-CoV-2. The diagnostic system is miniaturized (10 cm [height] \u00d7 9 cm [width] \u00d7 5.5 cm [length]) and lightweight (320 g), which can be operated with a portable battery and a smartphone. Once a liquid sample was loaded into an integrated microfluidic chip, a series of sample lysis, nucleic amplification, and real-time monitoring of the fluorescent signals of amplicons were automatically performed. Four reaction chambers were patterned on the chip, targeting As1e, N, E genes and a negative control, so multiple genes of SARS-CoV-2 could be simultaneously analyzed. The fluorescence intensities in each chamber were measured by a CMOS camera upon excitation with a 488 nm LED light source. The recorded data were processed by a microprocessor inside the IoT-based POC device and transferred and displayed on the wirelessly connected smartphone in real-time. The positive results could be obtained using three primer sets of SARS-CoV-2 with a limit of detection of 2 \u00d7 10^1 genome copies\/\u03bcL, and the clinical sample of SARS-CoV-2 was successfully analyzed with high sensitivity and accuracy. Our platform could provide an advanced molecular diagnostic tool to test SARS-CoV-2 anytime and anywhere.","lamp_id":[{"id":"LPB00447","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00448","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00449","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":236,"pmid":32239445,"title":"Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2","year":2020,"journal":"Virologica Sinica","authors":"Renfei Lu, Xiuming Wu, Zhenzhou Wan, Yingxue Li, Lulu Zuo, Jianru Qin, Xia Jin, Chiyu Zhang","doi":"10.1007\/s12250-020-00218-1","country":"China","institute":"Nantong Third Hospital Affiliated to Nantong University","deparment":"Clinical Laboratory","abstract":"No abstract available.","lamp_id":[{"id":"LPB00450","pathogen":"SARS-CoV-2","target":"RdRp"}]},{"id":237,"pmid":34771026,"title":"Robust Saliva-Based RNA Extraction-Free One-Step Nucleic Acid Amplification Test for Mass SARS-CoV-2 Monitoring","year":2021,"journal":"Molecules","authors":"Eva Rajh, Tina \u0160ket, Arne Praznik, Petra Su\u0161jan, Alenka \u0160mid, Dunja Urban\u010di\u010d, Irena Mlinari\u010d-Ra\u0161\u010dan, Polona Kogov\u0161ek, Tina Dem\u0161ar, Mojca Milavec, Katarina Prosenc Trilar, \u017diga Jensterle, Mihaela Zidarn, Viktorija Tomi\u010d, Gabriele Turel, Tatjana Lejko-Zupanc, Roman Jerala, Mojca Ben\u010dina","doi":"10.3390\/molecules26216617","country":"Slovenia","institute":"National Institute of Chemistry","deparment":"Department of Synthetic Biology and Immunology","abstract":"Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.","lamp_id":[{"id":"LPB00451","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00452","pathogen":"SARS-CoV-2","target":"E"}]},{"id":238,"pmid":34789179,"title":"Two extraction-free reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2","year":2021,"journal":"BMC Infectious Diseases","authors":"Meng Yee Lai, Fatma Diyana Mohd Bukhari, Nur Zulaikha Zulkefli, Ilyiana Ismail, Nur Izati Mustapa, Tuan Suhaila Tuan Soh, Afifah Haji Hassan, Kalaiarasu M Peariasamy, Yee Leng Lee, Jeyanthi Suppiah, Ravindran Thayan, Yee Ling Lau","doi":"10.1186\/s12879-021-06876-0","country":"Malaysia","institute":"University of Malaya","deparment":"Department of Parasitology","abstract":"Background: Current assays for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rely on time consuming, costly and laboratory based methods for virus isolation, purification and removing inhibitors. To address this limitation, we propose a simple method for testing RNA from nasopharyngeal swab samples that bypasses the RNA purification step.\n\nMethods: In the current project, we have described two extraction-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of SARS-CoV-2 by using E gene and RdRp gene as the targets.\n\nResults: Here, results showed that reverse transcription loop-mediated isothermal amplification assays with 88.4% sensitive (95% CI: 74.9-96.1%) and 67.4% sensitive (95% CI: 51.5-80.9%) for E gene and RdRp gene, respectively.\n\nConclusion: Without the need of RNA purification, our developed RT-LAMP assays for direct detection of SARS-CoV-2 from nasopharyngeal swab samples could be turned into alternatives to qRT-PCR for rapid screening.","lamp_id":[{"id":"LPB00453","pathogen":"SARS-CoV-2","target":"RdRp"},{"id":"LPB00454","pathogen":"SARS-CoV-2","target":"E"}]},{"id":239,"pmid":34677342,"title":"Portable and Label-Free Quantitative Loop-Mediated Isothermal Amplification (LF-qLamp) for Reliable COVID-19 Diagnostics in Three Minutes of Reaction Time: Arduino-Based Detection System Assisted by a pH Microelectrode","year":2021,"journal":"Biosensors","authors":"Mario Mois\u00e9s Alvarez, Sergio Bravo-Gonz\u00e1lez, Everardo Gonz\u00e1lez-Gonz\u00e1lez, Grissel Trujillo-de Santiago","doi":"10.3390\/bios11100386","country":"Mexico","institute":"Tecnologico de Monterrey\/Tecnologico de Monterrey","deparment":"Centro de Biotecnolog\u00eda-FEMSA\/Departamento de Bioingenier\u00eda","abstract":"Loop-mediated isothermal amplification (LAMP) has been recently studied as an alternative method for cost-effective diagnostics in the context of the current COVID-19 pandemic. Recent reports document that LAMP-based diagnostic methods have a comparable sensitivity and specificity to that of RT-qPCR. We report the use of a portable Arduino-based LAMP-based amplification system assisted by pH microelectrodes for the accurate and reliable diagnosis of SARS-CoV-2 during the first 3 min of the amplification reaction. We show that this simple system enables a straightforward discrimination between samples containing or not containing artificial SARS-CoV-2 genetic material in the range of 10 to 10,000 copies per 50 \u00b5L of reaction mix. We also spiked saliva samples with SARS-CoV-2 synthetic material and corroborated that the LAMP reaction can be successfully monitored in real time using microelectrodes in saliva samples as well. These results may have profound implications for the design of real-time and portable quantitative systems for the reliable detection of viral pathogens including SARS-CoV-2.","lamp_id":[{"id":"LPB00455","pathogen":"SARS-CoV-2","target":"N"}]},{"id":240,"pmid":34376716,"title":"Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection","year":2021,"journal":"Scientific Reports","authors":"Boon Lim, Jeremy Ratcliff, Dorota A Nawrot, Yejiong Yu, Harshmeena R Sanghani, Chia-Chen Hsu, Leon Peto, Simon Evans, Susanne H Hodgson, Aikaterini Skeva, Maria Adam, Maria Panopoulou, Christos E Zois, Katy Poncin, Sridhar R Vasudevan, Siqi Dai, Shuai Ren, Hong Chang, Zhanfeng Cui, Peter Simmonds, Wei E Huang, Monique I Andersson","doi":"10.1038\/s41598-021-95607-1","country":"United Kingdom","institute":"University of Oxford\/Institute of Biomedical Engineering","deparment":"Department of Engineering Science\/Nuffield Department of Medicine\/Department of Pharmacology","abstract":"We have optimised a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from extracted RNA for clinical application. We improved the stability and reliability of the RT-LAMP assay by the addition of a temperature-dependent switch oligonucleotide to reduce self- or off-target amplification. We then developed freeze-dried master mix for single step RT-LAMP reaction, simplifying the operation for end users and improving long-term storage and transportation. The assay can detect as low as 13 copies of SARS-CoV2 RNA per reaction (25-\u03bcL). Cross reactivity with other human coronaviruses was not observed. We have applied the new RT-LAMP assay for testing clinical extracted RNA samples extracted from swabs of 72 patients in the UK and 126 samples from Greece and demonstrated the overall sensitivity of 90.2% (95% CI 83.8-94.7%) and specificity of 92.4% (95% CI 83.2-97.5%). Among 115 positive samples which Ct values were less than 34, the RT-LAMP assay was able to detect 110 of them with 95.6% sensitivity. The specificity was 100% when RNA elution used RNase-free water. The outcome of RT-LAMP can be reported by both colorimetric detection and quantifiable fluorescent reading. Objective measures with a digitized reading data flow would allow for the sharing of results for local or national surveillance.","lamp_id":[{"id":"LPB00456","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":241,"pmid":34677325,"title":"A Portable RT-LAMP\/CRISPR Machine for Rapid COVID-19 Screening","year":2021,"journal":"Biosensors","authors":"Meysam Rezaei, Sajad Razavi Bazaz, Dorsa Morshedi Rad, Olga Shimoni, Dayong Jin, William Rawlinson, Majid Ebrahimi Warkiani","doi":"10.3390\/bios11100369","country":"Australia","institute":"University of Technology Sydney\/Institute for Biomedical Materials & Devices (IBMD)\/Genea","deparment":"School of Biomedical Engineering","abstract":"The COVID-19 pandemic has changed people's lives and has brought society to a sudden standstill, with lockdowns and social distancing as the preferred preventative measures. To lift these measurements and reduce society's burden, developing an easy-to-use, rapid, and portable system to detect SARS-CoV-2 is mandatory. To this end, we developed a portable and semi-automated device for SARS-CoV-2 detection based on reverse transcription loop-mediated isothermal amplification followed by a CRISPR\/Cas12a reaction. The device contains a heater element mounted on a printed circuit board, a cooler fan, a proportional integral derivative controller to control the temperature, and designated areas for 0.2 mL Eppendorf\u00ae PCR tubes. Our system has a limit of detection of 35 copies of the virus per microliter, which is significant and has the capability of being used in crisis centers, mobile laboratories, remote locations, or airports to diagnose individuals infected with SARS-CoV-2. We believe the current methodology that we have implemented in this article is beneficial for the early screening of infectious diseases, in which fast screening with high accuracy is necessary.","lamp_id":[{"id":"LPB00457","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00472","pathogen":"SARS-CoV-2","target":"N"}]},{"id":242,"pmid":34424659,"title":"SARS-CoV-2 Point-of-Care (POC) Diagnosis Based on Commercial Pregnancy Test Strips and a Palm-Size Microfluidic Device","year":2021,"journal":"Analytical Chemistry","authors":"Meiting Yang, Yidan Tang, Lijuan Qi, Sicai Zhang, Yichen Liu, Baiyang Lu, Jiaxue Yu, Kun Zhu, Bingling Li, Yan Du","doi":"10.1021\/acs.analchem.1c01829","country":"P. R. China","institute":"Changchun Institute of Applied Chemistry","deparment":"State Key Laboratory of Electroanalytical Chemistry","abstract":"Coronavirus diseases such as the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pose serious threats. Portable and accurate nucleic acid detection is still an urgent need to achieve on-site virus screening and timely infection control. Herein, we have developed an on-site, semiautomatic detection system, aiming at simultaneously overcoming the shortcomings suffered by various commercially available assays, such as low accuracy, poor portability, instrument dependency, and labor intensity. Ultrasensitive isothermal amplification [i.e., reverse transcription loop-mediated isothermal amplification (RT-LAMP)] was applied to generate intensified SARS-CoV-2 RNA signals, which were then transduced to portable commercial pregnancy test strips (PTSs) via ultraspecific human chorionic gonadotropin (hCG)-conjugated toehold-mediated strand exchange (TMSE) probes (hCG-P). The entire detection was integrated into a four-channel, palm-size microfluidic device, named the microfluidic point-of-care (POC) diagnosis system based on the PTS (MPSP) detection system. It provides rapid, cost-effective, and sensitive detection, of which the lowest concentration of detection was 0.5 copy\/\u03bcL of SARS-CoV-2 RNA, regardless of the presence of other similar viruses, even highly similar severe acute respiratory syndrome coronavirus (SARS-CoV). The successful detection of the authentic samples from different resources evaluated the practical application. The commercial PTS provides a colorimetric visible signal, which is instrument- and optimization-free. Therefore, this MPSP system can be immediately used for SARS-CoV-2 emergency detection, and it is worthy of further optimization to achieve full automation and detection for other infectious diseases.","lamp_id":[{"id":"LPB00458","pathogen":"SARS-CoV-2","target":"M"},{"id":"LPB00459","pathogen":"SARS-CoV-2","target":"N"}]},{"id":243,"pmid":34650067,"title":"Development, evaluation of the PNA RT-LAMP assay for rapid molecular detection of SARS-CoV-2","year":2021,"journal":"Scientific Reports","authors":"Chinbayar Bat-Ochir, Yeon-Sook Kim, Han Gyeul Kim, Si Seok Lee, Han Woo Lee, Hee Kyung Park","doi":"10.1038\/s41598-021-00041-y","country":"Korea","institute":"Seasun Biomaterials\/Chungnam National University School of Medicine","deparment":"Department of Internal Medicine","abstract":"Dual-labeled PNA probe used RT-LAMP molecular rapid assay targeting SARS-CoV-2 ORF1ab and N genes was developed, and the analytical, clinical performances for detection of SARS-CoV-2 RNA extracted from clinical nasopharyngeal swab specimens were evaluated in this study. Data showed that this assay is highly specific for SARS-CoV-2, and the absolute detection limit is 1 genomic copy per microliter of viral RNA which can be considered to be comparable to gold-standard molecular diagnostic method real-time reverse transcriptase PCR. Both clinical sensitivity and specificity against a commercial real-time RT-PCR assay were determined as identical. In conclusion, the PNA RT-LAMP assay showed high analytical and clinical accuracy which are identical to real-time RT-PCR which has been routinely used for the detection of SARS-CoV-2.","lamp_id":[{"id":"LPB00460","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00461","pathogen":"SARS-CoV-2","target":"N"}]},{"id":244,"pmid":34725400,"title":"A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test","year":2021,"journal":"Scientific Reports","authors":"Jonas Schmidt, Sandro Berghaus, Frithjof Blessing, Folker Wenzel, Holger Herbeck, Josef Blessing, Peter Schierack, Stefan R\u00f6diger, Dirk Roggenbuck","doi":"10.1038\/s41598-021-00827-0","country":"Germany","institute":"Institute for Laboratory Medicine\/Furtwangen University\/Brandenburg University of Technology Cottbus-Senftenberg","deparment":"Faculty of Medical and Life Sciences\/Faculty Environment and Natural Sciences","abstract":"Shortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen's kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar's test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories.","lamp_id":[{"id":"LPB00462","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00463","pathogen":"SARS-CoV-2","target":"E"}]},{"id":245,"pmid":34741956,"title":"Rapid detection of novel coronavirus SARS-CoV-2 by RT-LAMP coupled solid-state nanopores","year":2022,"journal":"Biosensors and Bioelectronics","authors":"Zifan Tang, Reza Nouri, Ming Dong, Jianbo Yang, Wallace Greene, Yusheng Zhu, Michele Yon, Meera Surendran Nair, Suresh V Kuchipudi, Weihua Guan","doi":"10.1016\/j.bios.2021.113759","country":"USA","institute":"Pennsylvania State University","deparment":"Department of Electrical Engineering","abstract":"The current pandemic of COVID-19 caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) has raised significant public health concerns. Rapid and accurate testing of SARS-CoV-2 is urgently needed for early detection and control of the disease spread. Here, we present an RT-LAMP coupled glass nanopore digital counting method for rapid detection of SARS-CoV-2. We validated and compared two one-pot RT-LAMP assays targeting nucleocapsid (N) and envelop (E) genes. The nucleocapsid assay was adopted due to its quick time to positive and better copy number sensitivity. For qualitative positive\/negative classification of a testing sample, we used the glass nanopore to digitally count the RT-LAMP amplicons and benchmarked the event rate with a threshold. Due to its intrinsic single molecule sensitivity, nanopore sensors could capture the amplification dynamics more rapidly (quick time to positive). We validated our RT-LAMP coupled glass nanopore digital counting method for SARS-CoV-2 detection by using both spiked saliva samples and COVID-19 clinical nasopharyngeal swab samples. The results obtained showed excellent agreement with the gold standard RT-PCR assay. With its integration capability, the electronic nanopore digital counting platform has significant potential to provide a rapid, sensitive, and specific point-of-care assay for SARS-CoV-2.","lamp_id":[{"id":"LPB00464","pathogen":"SARS-CoV-2","target":"N"}]},{"id":246,"pmid":34308531,"title":"RT-LAMP: A Cheaper, Simpler and Faster Alternative for the Detection of SARS-CoV-2 in Wastewater","year":2021,"journal":"Food and Environmental Virology","authors":"Isaac Dennis Amoah, Nonsikelelo Precios Mthethwa, Leanne Pillay, Nashia Deepnarain, Kriveshin Pillay, Oluyemi Olatunji Awolusi, Sheena Kumari, Faizal Bux","doi":"10.1007\/s12560-021-09489-7","country":"South Africa","institute":"Durban University of Technology","deparment":"Institute for Water and Wastewater Technology","abstract":"Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has the potential to become a cheaper and faster option for monitoring COVID-19 infections through wastewater-based epidemiology. However, its application in COVID-19 surveillance has been limited to clinical testing only. We present in this paper two optimized RT-LAMP protocols based on colour change and fluorescence detection and application of these protocols for wastewater monitoring from four wastewater treatment plants over 4 weeks. The optimized RT-LAMP protocols have a limit of detection of 10 copies\/25 \u00b5l reaction with positive amplification within 35 minutes. Over the 4 weeks of monitoring, the colorimetric protocol detected a prevalence of 12.5%, when 1 \u00b5l of extracted RNA with 92.7(\u00b1 28.2) ng\/\u00b5l concentration was analysed. When the RNA template was increased by fivefold, the prevalence increased to 44%. The fluorescent RT-LAMP had a prevalence of 31% and 47% for starting templates of 92.7(\u00b1 28.2) ng\/\u00b5l and 480(\u00b1 134.5) ng\/\u00b5l of the extracted RNA, respectively. All samples were positive for SARS-CoV-2 when analysed with droplet digital PCR, with viral loads ranging from 18.1 to 195.6 gc\/ml of wastewater. The RT-ddPCR, therefore, confirms the presence of the viral RNA in the wastewater samples, albeit at low concentrations. Additionally, the RT-LAMP protocols positively detected SARS-CoV-2 in wastewater samples with copies as low as 20.7 gc\/ml. The results obtained in our study show the potential application of RT-LAMP for the detection of SARS-CoV-2 in wastewater, which could provide a cheaper and faster alternative to RT-qPCR or RT-ddPCR for wastewater-based epidemiological monitoring of COVID-19 and other viral infections.","lamp_id":[{"id":"LPB00465","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00466","pathogen":"SARS-CoV-2","target":"N"}]},{"id":247,"pmid":35290826,"title":"A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern","year":2022,"journal":"EBioMedicine","authors":"Long T Nguyen, Nicolas C Macaluso, Brianna L M Pizzano, Melanie N Cash, Jan Spacek, Jan Karasek, Megan R Miller, John A Lednicky, Rhoel R Dinglasan, Marco Salemi, Piyush K Jain","doi":"10.1016\/j.ebiom.2022.103926","country":"USA","institute":"University of Florida","deparment":"Department of Chemical Engineering","abstract":"Background: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR\/Cas (Clustered Regularly Interspaced Short Palindromic Repeats\/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs.\n\nMethods: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs).\n\nFindings: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92\u00b78% sensitivity, 99\u00b74% specificity, and 96\u00b77% accuracy within 10-30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99\u00b71% and 95\u00b71%, respectively. Interestingly, for samples with high viral load (Ct value \u2264 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals.\n\nInterpretation: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19.","lamp_id":[{"id":"LPB00467","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00468","pathogen":"SARS-CoV-2 (Alpha)","target":"S"},{"id":"LPB00469","pathogen":"SARS-CoV-2 (Beta)","target":"S"},{"id":"LPB00470","pathogen":"SARS-CoV-2 (Delta)","target":"S"},{"id":"LPB00471","pathogen":"SARS-CoV-2 (Gamma)","target":"ORF8-N"},{"id":"LPB00505","pathogen":"SARS-CoV-2 (Omicron)","target":"S"}]},{"id":248,"pmid":32837510,"title":"A fully automated centrifugal microfluidic system for sample-to-answer viral nucleic acid testing","year":2020,"journal":"Science China Chemistry","authors":"Fei Tian, Chao Liu, Jinqi Deng, Ziwei Han, Lu Zhang, Qinghua Chen, Jiashu Sun","doi":"10.1007\/s11426-020-9800-6","country":"China","institute":"National Center for Nanoscience and Technology\/University of Chinese Academy of Sciences","deparment":"CAS Key Laboratory of Standardization and Measurement of Nanotechnolog","abstract":"The outbreak of virus-induced infectious diseases poses a global public-health challenge. Nucleic acid amplification testing (NAAT) enables early detection of pandemic viruses and plays a vital role in preventing onward transmission. However, the requirement of skilled operators, expensive instrumentation, and biosafety laboratories has hindered the use of NAAT for screening and diagnosis of suspected patients. Here we report development of a fully automated centrifugal microfluidic system with sample-in-answer-out capability for sensitive, specific, and rapid viral nucleic acid testing. The release of nucleic acids and the subsequent reverse transcription loop-mediated isothermal amplification (RT-LAMP) were integrated into the reaction units of a microfluidic disc. The whole processing steps such as injection of reagents, fluid actuation by rotation, heating and temperature control, and detection of fluorescence signals were carried out automatically by a customized instrument. We validate the centrifugal microfluidic system using oropharyngeal swab samples spiked with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) armored RNA particles. The estimated limit of detection for armored RNA particles is 2 copies per reaction, the throughput is 21 reactions per disc, and the assay sample-to-answer time is approximately 70 min. This enclosed and automated microfluidic system efficiently avoids viral contamination of aerosol, and can be readily adapted for virus detection outside the diagnostic laboratory.","lamp_id":[{"id":"LPB00473","pathogen":"SARS-CoV-2","target":"N"}]},{"id":249,"pmid":33493900,"title":"Carrying out pseudo dual nucleic acid detection from sample to visual result in a polypropylene bag with CRISPR\/Cas12a","year":2021,"journal":"Biosensors and Bioelectronics","authors":"Hui Wu, Yanju Chen, Ya Shi, Liu Wang, Mengyao Zhang, Jian Wu, Huan Chen","doi":"10.1016\/j.bios.2021.113001","country":"China","institute":"Zhejiang University","deparment":"College of Biosystems Engineering and Food Science","abstract":"Amplification-based nucleic acid detection is widely employed in food safety, medical diagnosis and environment monitoring. However, conventional nucleic acid analysis has to be carried out in laboratories because of requiring expensive instruments and trained personnel. If people could do nucleic acid detection at home by themselves, the application of nucleic acid detection would be greatly accelerated. We herein reported a polypropylene (PP) bag-based method for convenient detection of nucleic acids in the oil-sealed space. The PP bag has three chambers which are responsible for lysis, washing and amplification\/detection, respectively. After adding sample, nucleic acids are adsorbed on magnetic particles (MPs) and moved into these three chambers successively through immiscible oil channel by an external magnet. Combined with isothermal amplification, the PP bag can be incubated in a water bath or milk warmer and acted as a reaction tube. With highly specific CRISPR technology, Salmonella typhimurium (St) and SARS-CoV-2 can be visually detected in these PP bags within 1 h, indicating its potential household application. To further improve the reliability of nucleic acid testing at home, a logic decision method is introduced by detecting both target and endogenous reference gene. Positive\/negative\/invalid detection result can be obtained by chronologically adding the CRISPR reagents of target and endogenous reference gene. We anticipate that this PP bag can provide a novel toolkit for nucleic acid detection in people's daily life.","lamp_id":[{"id":"LPB00474","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":250,"pmid":34441334,"title":"A Novel Saliva RT-LAMP Workflow for Rapid Identification of COVID-19 Cases and Restraining Viral Spread","year":2021,"journal":"Diagnostics","authors":"Gerson Shigeru Kobayashi, Luciano Abreu Brito, Danielle de Paula Moreira, Angela May Suzuki, Gabriella Shih Ping Hsia, Lylyan Fragoso Pimentel, Ana Paula Barreto de Paiva, Carolina Regoli Dias, Naila Cristina Vila\u00e7a Louren\u00e7o, Beatriz Araujo Oliveira, Erika Regina Manuli, Marcelo Andreetta Corral, Natale Cava\u00e7ana, Miguel Mitne-Neto, Maria Mirtes Sales, Luiz Phellipe Dell' Aquila, Alvaro Razuk Filho, Eduardo Fagundes Parrillo, Maria C\u00e1ssia Mendes-Corr\u00eaa, Ester Cerdeira Sabino, Silvia Figueiredo Costa, Fabio Eudes Leal, Germ\u00e1n Gustavo Sgro, Chuck Shaker Farah, Mayana Zatz, Maria Rita Passos-Bueno","doi":"10.3390\/diagnostics11081400","country":"Brazil","institute":"Instituto de Bioci\u00eancias, Universidade de S\u00e3o Paulo (USP)","deparment":"Centro de Pesquisa Sobre o Genoma Humano e C\u00e9lulas-Tronco (HUG-CELL)","abstract":"Rapid diagnostics is pivotal to curb SARS-CoV-2 transmission, and saliva has emerged as a practical alternative to naso\/oropharyngeal (NOP) specimens. We aimed to develop a direct RT-LAMP (reverse transcription loop-mediated isothermal amplification) workflow for viral detection in saliva, and to provide more information regarding its potential in curbing COVID-19 transmission. Clinical and contrived specimens were used to optimize formulations and sample processing protocols. Salivary viral load was determined in symptomatic patients to evaluate the clinical performance of the test and to characterize saliva based on age, gender and time from onset of symptoms. Our workflow achieved an overall sensitivity of 77.2% (n = 90), with 93.2% sensitivity, 97% specificity, and 0.895 Kappa for specimens containing >10^2 copies\/\u03bcL (n = 77). Further analyses in saliva showed that viral load peaks in the first days of symptoms and decreases afterwards, and that viral load is ~10 times lower in females compared to males, and declines following symptom onset. NOP RT-PCR data did not yield relevant associations. This work suggests that saliva reflects the transmission dynamics better than NOP specimens, and reveals gender differences that may reflect higher transmission by males. This saliva RT-LAMP workflow can be applied to track viral spread and, to maximize detection, testing should be performed immediately after symptoms are presented, especially in females.","lamp_id":[{"id":"LPB00475","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00476","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":251,"pmid":35089353,"title":"Assessment of a Smartphone-Based Loop-Mediated Isothermal Amplification Assay for Detection of SARS-CoV-2 and Influenza Viruses","year":2022,"journal":"JAMA Network Open","authors":"Douglas M Heithoff, Lucien Barnes 5th, Scott P Mahan, Gary N Fox, Katherine E Arn, Sarah J Ettinger, Andrew M Bishop, Lynn N Fitzgibbons, Jeffrey C Fried, David A Low, Charles E Samuel, Michael J Mahan","doi":"10.1001\/jamanetworkopen.2021.45669","country":"USA","institute":"University of California","deparment":"Department of Molecular, Cellular, and Developmental Biology\/Institute for Collaborative Biotechnologies","abstract":"Importance: A critical need exists in low-income and middle-income countries for low-cost, low-tech, yet highly reliable and scalable testing for SARS-CoV-2 virus that is robust against circulating variants.\n\nObjective: To assess whether a smartphone-based assay is suitable for SARS-CoV-2 and influenza virus testing without requiring specialized equipment, accessory devices, or custom reagents.\n\nDesign, setting, and participants: This cohort study enrolled 2 subgroups of participants (symptomatic and asymptomatic) at Santa Barbara Cottage Hospital. The symptomatic group consisted of 20 recruited patients who tested positive for SARS-CoV-2 with symptoms; 30 asymptomatic patients were recruited from the same community, through negative admission screening tests for SARS-CoV-2. The smartphone-based real-time loop-mediated isothermal amplification (smaRT-LAMP) was first optimized for analysis of human saliva samples spiked with either SARS-CoV-2 or influenza A or B virus; these results then were compared with those obtained by side-by-side analysis of spiked samples using the Centers for Disease Control and Prevention (CDC) criterion-standard reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay. Next, both assays were used to test for SARS-CoV-2 and influenza viruses present in blinded clinical saliva samples obtained from 50 hospitalized patients. Statistical analysis was performed from May to June 2021.\n\nExposures: Testing for SARS-CoV-2 and influenza A and B viruses.\n\nMain outcomes and measures: SARS-CoV-2 and influenza infection status and quantitative viral load were determined.\n\nResults: Among the 50 eligible participants with no prior SARS-CoV-2 infection included in the study, 29 were men. The mean age was 57 years (range, 21 to 93 years). SmaRT-LAMP exhibited 100% concordance (50 of 50 patient samples) with the CDC criterion-standard diagnostic for SARS-CoV-2 sensitivity (20 of 20 positive and 30 of 30 negative) and for quantitative detection of viral load. This platform also met the CDC criterion standard for detection of clinically similar influenza A and B viruses in spiked saliva samples (n = 20), and in saliva samples from hospitalized patients (50 of 50 negative). The smartphone-based LAMP assay was rapid (25 minutes), sensitive (1000 copies\/mL), low-cost (<$7\/test), and scalable (96 samples\/phone).\n\nConclusions and relevance: In this cohort study of saliva samples from patients, the smartphone-based LAMP assay detected SARS-CoV-2 infection and exhibited concordance with RT-qPCR tests. These findings suggest that this tool could be adapted in response to novel CoV-2 variants and other pathogens with pandemic potential including influenza and may be useful in settings with limited resources.","lamp_id":[{"id":"LPB00477","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00478","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00479","pathogen":"Influenza A virus (H1N1, H3N2)","target":"PB1"},{"id":"LPB00480","pathogen":"Influenza B virus","target":"M1"},{"id":"LPB00481","pathogen":"Influenza B virus","target":"NS1"},{"id":"LPB00482","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00483","pathogen":"Influenza A virus (H1N1, H3N2)","target":"M1"},{"id":"LPB00485","pathogen":"SARS-CoV-2","target":"N"}]},{"id":252,"pmid":35043022,"title":"Optimization of loop-mediated isothermal amplification (LAMP) assay for robust visualization in SARS-CoV-2 and emerging variants diagnosis","year":2022,"journal":"Chemical Engineering Science","authors":"Zhen Luo, Chunhong Ye, Heng Xiao, Jialing Yin, Yicong Liang, Zhihui Ruan, Danju Luo, Daolong Gao, Qiuping Tan, Yongkui Li, Qiwei Zhang, Weiyong Liu, Jianguo Wu","doi":"10.1016\/j.ces.2022.117430","country":"China","institute":"Institute of Medical Microbiology\/Foshan Institute of Medical Microbiology","deparment":"Guangdong Provincial Key Laboratory of Virology","abstract":"Loop-mediated isothermal amplification (LAMP) is widely used in detection of pathogenic microorganisms including SARS-CoV-2. However, the performance of LAMP assay needs further exploration in the emerging SARS-CoV-2 variants test. Here, we design serials of primers and select an optimal set for LAMP-based on SARS-CoV-2 N gene for a robust and visual assay in SARS-CoV-2 diagnosis. The limit of detectable template reaches 10 copies of N gene per 25 \u03bcL reaction at isothermal 58\u2103 within 40 min. Importantly, the primers for LAMP assay locate at 12 to 213 nt of N gene, a highly conservative region, which serves as a compatible test in emerging SARS-CoV-2 variants. Comparison to a commercial qPCR assay, this LAMP assay exerts the high viability in diagnosis of 41 clinical samples. Our study optimizes an advantageous LAMP assay for colorimetric detection of SARS-CoV-2 and emerging variants, which is hopeful to be a promising test in COVID-19 surveillance.","lamp_id":[{"id":"LPB00486","pathogen":"SARS-CoV-2","target":"N"}]},{"id":253,"pmid":35044487,"title":"Lateral flow-based nucleic acid detection of SARS-CoV-2 using enzymatic incorporation of biotin-labeled dUTP for POCT use","year":2022,"journal":"Analytical and Bioanalytical Chemistry","authors":"Saloni Agarwal, Christian Warmt, Joerg Henkel, Livia Schrick, Andreas Nitsche, Frank F Bier","doi":"10.1007\/s00216-022-03880-4","country":"Germany","institute":"University of Potsdam","deparment":"Institute for Biochemistry and Biology","abstract":"The degree of detrimental effects inflicted on mankind by the COVID-19 pandemic increased the need to develop ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable) POCT (point of care testing) to overcome the current and any future pandemics. Much effort in research and development is currently advancing the progress to overcome the diagnostic pressure built up by emerging new pathogens. LAMP (loop-mediated isothermal amplification) is a well-researched isothermal technique for specific nucleic acid amplification which can be combined with a highly sensitive immunochromatographic readout via lateral flow assays (LFA). Here we discuss LAMP-LFA robustness, sensitivity, and specificity for SARS-CoV-2 N-gene detection in cDNA and clinical swab-extracted RNA samples. The LFA readout is designed to produce highly specific results by incorporation of biotin and FITC labels to 11-dUTP and LF (loop forming forward) primer, respectively. The LAMP-LFA assay was established using cDNA for N-gene with an accuracy of 95.65%. To validate the study, 82 SARS-CoV-2-positive RNA samples were tested. Reverse transcriptase (RT)-LAMP-LFA was positive for the RNA samples with an accuracy of 81.66%; SARS-CoV-2 viral RNA was detected by RT-LAMP-LFA for as low as CT-33. Our method reduced the detection time to 15 min and indicates therefore that RT-LAMP in combination with LFA represents a promising nucleic acid biosensing POCT platform that combines with smartphone based semi-quantitative data analysis.","lamp_id":[{"id":"LPB00487","pathogen":"SARS-CoV-2","target":"N"}]},{"id":254,"pmid":35293849,"title":"RT-LAMP assay for rapid detection of the R203M mutation in SARS-CoV-2 Delta variant","year":2022,"journal":"Emerging Microbes & Infections","authors":"Jianing Yang, Xuejiao Hu, Wenzhuo Wang, Yujing Yang, Xinqiang Zhang, Wei Fang, Lei Zhang, Shan Li, Bing Gu","doi":"10.1080\/22221751.2022.2054368","country":"China","institute":"South China University of Technology","deparment":"MOE International Joint Laboratory for Synthetic Biology and Medicines","abstract":"The highly infectious Delta variant strain of SARS-CoV-2 remains globally dominant and undermines COVID-19 vaccines. Rapid detection of the Delta variant is crucial for the identification and quarantine of infected individuals. In this study, our aim was to design and validate a genotyping RT-LAMP method to detect Delta variants specifically. R203M in the N gene of SARS-CoV-2 was chosen as the Delta variant-specific mutation for genotyping. To target the R203M-harboring region and the conserved sequence of the N gene, two sets of primers were designed, and a Cq (quantification cycle) ratio-based RT-LAMP for SARS-CoV-2 and R203M detection was developed by analyzing the significant discrepancy in amplification efficiency of the two sets of primers. We validated the RT-LAMP method on 498 clinical specimens in parallel with RT-qPCR, and 84 Delta variants from 198 positive samples were determined by sequencing. Compared with traditional RT-qPCR analyses, RT-LAMP appears to be 100% accurate in detecting SARS-CoV-2 clinical samples. RT-LAMP has a good ability to distinguish between Delta and non-Delta variants under a Cq ratio threshold of 1.80. Furthermore, the AUC (area under the curve) of this method was 1.00; the sensitivity, specificity and accuracy were all 100%. In summary, we have proposed a rapid, accurate and cost-effective RT-LAMP method to detect SARS-CoV-2 and Delta variants, which may facilitate the surveillance of COVID-19.","lamp_id":[{"id":"LPB00488","pathogen":"SARS-CoV-2 (Delta)","target":"N"},{"id":"LPB00489","pathogen":"SARS-CoV-2","target":"N"}]},{"id":255,"pmid":35256137,"title":"A point-of-care SARS-CoV-2 test based on reverse transcription loop-mediated isothermal amplification without RNA extraction with diagnostic performance same as RT-PCR","year":2022,"journal":"Analytica Chimica Acta","authors":"Nelson Odiwuor, Jin Xiong, Faith Ogolla, Wei Hong, Xiaohong Li, Fazal Mehmood Khan, Nuo Wang, Junping Yu, Hongping Wei","doi":"10.1016\/j.aca.2022.339590","country":"China\/Kenya","institute":"Wuhan Institute of Virology\/University of Chinese Academy of Sciences\/Sino-Africa Joint Research Centre","deparment":"CAS Key Laboratory of Special Pathogens and Biosafety","abstract":"The global public health crisis and economic losses resulting from the current novel coronavirus disease (COVID-19) pandemic have been dire. The most used real-time reverse transcription polymerase chain reaction (RT-PCR) method needs expensive equipment, technical expertise, and a long turnaround time. Therefore, there is a need for a rapid, accurate, and alternative technique of diagnosis that is deployable at resource-poor settings like point-of-care. This study combines heat deactivation and a novel mechanical lysis method by bead beating for quick and simple sample preparation. Then, using an optimized reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to target genes encoding the open reading frame 8 (ORF8), spike and nucleocapsid proteins of the novel coronavirus, SARS-CoV-2. The test results can be read simultaneously in fluorometric and colorimetric readouts within 40 min from sample collection. We also calibrated a template transfer tool to simplify sample addition into LAMP reactions when pipetting skills are needed. Most importantly, validation of the direct RT-LAMP system based on multiplexing primers S1:ORF8 in a ratio (1:0.8) using 143 patients' nasopharyngeal swab samples showed a diagnostic performance of 99.30% accuracy, with 98.81% sensitivity and 100% selectivity, compared to commercial RT-PCR kits. Since our workflow does not rely on RNA extraction and purification, the time-to-result is two times faster than other workflows with FDA emergency use authorization. Considering all its strengths: speed, simplicity, accuracy and extraction-free, the system can be useful for optimal point-of-care testing of COVID-19.","lamp_id":[{"id":"LPB00490","pathogen":"SARS-CoV-2","target":"ORF8"},{"id":"LPB00491","pathogen":"SARS-CoV-2","target":"S"}]},{"id":256,"pmid":35251932,"title":"Loop-mediated isothermal amplification-based electrochemical sensor for detecting SARS-CoV-2 in wastewater samples","year":2022,"journal":"Journal of Environmental Chemical Engineering","authors":"Roberto G Ram\u00edrez-Chavarr\u00eda, Elizabeth Castillo-Villanueva, Bryan E Alvarez-Serna, Juli\u00e1n Carrillo-Reyes, Rosa Mar\u00eda Ram\u00edrez-Zamora, Germ\u00e1n Buitr\u00f3n, Luis Alvarez-Icaza","doi":"10.1016\/j.jece.2022.107488","country":"M\u00e9xico","institute":"Universidad Nacional Aut\u00f3noma de M\u00e9xico","deparment":"Instituto de Ingenier\u00eda","abstract":"The current pandemic COVID-19 caused by the coronavirus SARS-CoV-2, has generated different economic, social and public health problems. Moreover, wastewater-based epidemiology could be a predictor of the virus rate of spread to alert on new outbreaks. To assist in epidemiological surveillance, this work introduces a simple, low-cost and affordable electrochemical sensor to specifically detect N and ORF1ab genes of the SARS-CoV-2 genome. The proposed sensor works based on screen-printed electrodes acting as a disposable test strip, where the reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction takes place. Electrochemical detection relies upon methylene blue as a redox intercalator probe, to provide a diffusion-controlled current encoding the presence and concentration of RT-LAMP products, namely amplicons or double-stranded DNA. We test the performance of the sensor by testing real wastewater samples using end-point and time course measurements. Results show the ability of the electrochemical test strip to specifically detect and quantify RT-LAMP amplicons below to ~ 2.5 \u00d7 10^-6 ng\/\u03bcL exhibiting high reproducibility. In this sense, our RT-LAMP electrochemical sensor is an attractive, efficient and powerful tool for rapid and reliable wastewater-based epidemiology studies.","lamp_id":[{"id":"LPB00492","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00493","pathogen":"SARS-CoV-2","target":"N"}]},{"id":257,"pmid":35388102,"title":"SaliVISION: a rapid saliva-based COVID-19 screening and diagnostic test with high sensitivity and specificity","year":2022,"journal":"Scientific Reports","authors":"Samuel M DeFina, Jianhui Wang, Lei Yang, Han Zhou, Jennifer Adams, William Cushing, Beth Tuohy, Pei Hui, Chen Liu, Kien Pham","doi":"10.1038\/s41598-022-09718-4","country":"USA","institute":"Yale University","deparment":"Department of Pathology","abstract":"The Coronavirus disease 2019 (COVID-19) pandemic-caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)- has posed a global threat and presented with it a multitude of economic and public-health challenges. Establishing a reliable means of readily available, rapid diagnostic testing is of paramount importance in halting the spread of COVID-19, as governments continue to ease lockdown restrictions. The current standard for laboratory testing utilizes reverse transcription quantitative polymerase chain reaction (RT-qPCR); however, this method presents clear limitations in requiring a longer run-time as well as reduced on-site testing capability. Therefore, we investigated the feasibility of a reverse transcription looped-mediated isothermal amplification (RT-LAMP)-based model of rapid COVID-19 diagnostic testing which allows for less invasive sample collection, named SaliVISION. This novel, two-step, RT-LAMP assay utilizes a customized multiplex primer set specifically targeting SARS-CoV-2 and a visual report system that is ready to interpret within 40 min from the start of sample processing and does not require a BSL-2 level testing environment or special laboratory equipment. When compared to the SalivaDirect and Thermo Fisher Scientific TaqPath RT-qPCR testing platforms, the respective sensitivities of the SaliVISION assay are 94.29% and 98.28% while assay specificity was 100% when compared to either testing platform. Our data illustrate a robust, rapid diagnostic assay in our novel RT-LAMP test design, with potential for greater testing throughput than is currently available through laboratory testing and increased on-site testing capability.","lamp_id":[{"id":"LPB00494","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00495","pathogen":"SARS-CoV-2","target":"N"}]},{"id":258,"pmid":35487969,"title":"A lyophilized colorimetric RT-LAMP test kit for rapid, low-cost, at-home molecular testing of SARS-CoV-2 and other pathogens","year":2022,"journal":"Scientific Reports","authors":"Xin Song, Felicity J Coulter, Ming Yang, Jessica L Smith, Fikadu G Tafesse, William B Messer, John H Reif","doi":"10.1038\/s41598-022-11144-5","country":"USA","institute":"Duke University","deparment":"Department of Electrical and Computer Engineering","abstract":"Access to fast and reliable nucleic acid testing continues to play a key role in controlling the COVID-19 pandemic, especially in the context of increased vaccine break-through risks due to new variants. We report a rapid, low-cost (~ 2 USD), simple-to-use nucleic acid test kit for self-administered at-home testing without lab instrumentation. The entire sample-to-answer workflow takes < 60 min, including noninvasive sample collection, one-step RNA preparation, reverse-transcription loop-mediated isothermal amplification (RT-LAMP) in a thermos, and direct visual inspection of a colorimetric test result. To facilitate long-term storage without cold-chain, a fast one-pot lyophilization protocol was developed to preserve all required biochemical reagents of the colorimetric RT-LAMP test in a single microtube. Notably, the lyophilized RT-LAMP assay demonstrated reduced false positives as well as enhanced tolerance to a wider range of incubation temperatures compared to solution-based RT-LAMP reactions. We validated our RT-LAMP assay using simulated infected samples, and detected a panel of SARS-CoV-2 variants with successful detection of all variants that were available to us at the time. With a simple change of the primer set, our lyophilized RT-LAMP home test can be easily adapted as a low-cost surveillance platform for other pathogens and infectious diseases of global public health importance.","lamp_id":[{"id":"LPB00496","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":259,"pmid":35472524,"title":"Colorimetric detection of SARS-CoV-2 by uracil-DNA glycosylase (UDG) reverse transcription loop-mediated isothermal amplification (RT-LAMP)","year":2022,"journal":"International Journal of Infectious Diseases","authors":"Meng Yee Lai , Fatma Diyana Mohd Bukhari, Nur Zulaikha Zulkefli, Ilyiana Ismail, Nur Izati Mustapa, Tuan Suhaila Tuan Soh, Afifah Haji Hassan, Kalaiarasu M Peariasamy, Yee Leng Lee, Jeyanthi Suppiah, Ravindran Thayan, Yee Ling Lau","doi":"10.1016\/j.ijid.2022.04.036","country":"Malaysia","institute":"Universiti Malaya","deparment":"Department of Parasitology","abstract":"Objectives: Preventing reverse transcription loop-mediated isothermal amplification (RT-LAMP) carryover contamination could be solved by adding deoxyuridine triphosphate (dUTP) and uracil-DNA glycosylase (UDG) into the reaction master mix.\n\nMethods: RNA was extracted from nasopharyngeal swab samples by a simple RNA extraction method.\n\nResults: Testing of 77 samples demonstrated 91.2% sensitivity (95% confidence interval [CI]: 78-98.2%) and 100% specificity (95% confidence interval: 92-100%) using UDG RT-LAMP.\n\nConclusion: This colorimetric UDG RT-LAMP is a simple-to-use, fast, and easy-to-interpret method, which could serve as an alternative for diagnosis of SARS-CoV-2 infection, especially in remote hospitals and laboratories with under-equipped medical facilities.","lamp_id":[{"id":"LPB00497","pathogen":"SARS-CoV-2","target":"N"}]},{"id":260,"pmid":35453896,"title":"A Rapid RT-LAMP Assay for SARS-CoV-2 with Colorimetric Detection Assisted by a Mobile Application","year":2022,"journal":"Diagnostics","authors":"Mar\u00eda Aurora Londono-Avendano, Gerardo Libreros, Lyda Osorio, Beatriz Parra","doi":"10.3390\/diagnostics12040848","country":"Colombia","institute":"Universidad del Valle","deparment":"Departamento de Microbiolog\u00eda","abstract":"Loop-mediated amplification has been promoted for SARS-CoV-2 screening, however, antigen tests are preferred in low-income countries and remote zones. Poor training in molecular biology, plus the need for RNA purification or reading instruments to overcome issues of sensitivity in colorimetric detection, are some of the reasons limiting the use of this technique. In this study, nasopharyngeal swabs, aspirates and saliva were amplified in an in-house LAMP assay and subject to colorimetric detection, achieved by the naked eye and by image analysis with a mobile application. Accuracy of detection by the naked eye ranged from 61-74% but improved to 75-86% when using the application. Sensitivity of the digital approach was 81% and specificity 83%, with poor positive predictive value, and acceptable negative predictive value. Additionally to the reported effect of some transport media's pH, the presence of mucus and warming up of reagents while setting up the reaction critically affected performance. Accuracy per type of sample was 55, 70 and 80%, for swabs, aspirates and saliva, respectively, suggesting potential to improve the test in saliva. This assay, carried out in a closed tube, reduces contamination, has few pipetting steps and requires minimal equipment. Strategies to improve performance and implications of the use this sort of colorimetric LAMP for massive testing are discussed.","lamp_id":[{"id":"LPB00498","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":261,"pmid":35396760,"title":"CLEVER assay: A visual and rapid RNA extraction-free detection of SARS-CoV-2 based on CRISPR-Cas integrated RT-LAMP technology","year":2022,"journal":"Journal of Applied Microbiology","authors":"Akansha Bhatt, Zeeshan Fatima, Munindra Ruwali, Chitra Seetharam Misra, Shyam Sunder Rangu, Devashish Rath, Ashok Rattan, Saif Hameed","doi":"10.1111\/jam.15571","country":"India","institute":"Amity University Haryana","deparment":"Amity Institute of Biotechnology","abstract":"Aim: The current scenario of COVID-19 pandemic has presented an almost insurmountable challenge even for the most sophisticated hospitals equipped with modern biomedical technology. There is an urgency to develop simple, fast and highly accurate methods for the rapid identification and isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients. To address the ongoing challenge, the present study offers a CLEVER assay (CRISPR-Cas integrated RT-LAMP Easy, Visual and Extraction-free RNA) which will allow RNA extraction-free method to visually diagnose COVID-19. RNA extraction is a major hurdle in preventing rapid and large-scale screening of samples particularly in low-resource regions because of the logistics and costs involved.\n\nMethod and result: Herein, the visual SARS-CoV-2 detection method consists of RNA extraction-free method directly utilizing the patient's nasopharyngeal and oropharyngeal samples for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Additionally, the assay also utilizes the integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12-based system using different guide RNAs of N, E and an internal control POP7 (human RNase P) genes along with visual detection via lateral flow readout-based dip sticks with unaided eye (~100 min). Overall, the clinical sensitivity and specificity of the CLEVER assay were 89.6% and 100%, respectively.\n\nConclusion: Together, our CLEVER assay offers a point-of-care tool with no equipment dependency and minimum technical expertise requirement for COVID-19 diagnosis.","lamp_id":[{"id":"LPB00499","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00500","pathogen":"SARS-CoV-2","target":"E"}]},{"id":262,"pmid":35192340,"title":"Multiplex, Real-Time, Point-of-care RT-LAMP for SARS-CoV-2 Detection Using the HFman Probe","year":2022,"journal":"ACS Sensors","authors":"Yajuan Dong, Yongjuan Zhao, Shenwei Li, Zhenzhou Wan, Renfei Lu, Xianguang Yang, Guoying Yu, Julien Reboud, Jonathan M Cooper, Zhengan Tian, Chiyu Zhang","doi":"10.1021\/acssensors.1c02079","country":"China","institute":"Henan Normal University","deparment":"College of Life Sciences","abstract":"Viral evolution impacts diagnostic test performance through the emergence of variants with sequences affecting the efficiency of primer binding. Such variants that evade detection by nucleic acid-based tests are subject to selective pressure, enabling them to spread more efficiently. Here, we report a variant-tolerant diagnostic test for SARS-CoV-2 using a loop-mediated isothermal nucleic acid-based amplification (LAMP) assay containing high-fidelity DNA polymerase and a high-fidelity DNA polymerase-medicated probe (HFman probe). In addition to demonstrating a high tolerance to variable SARS-CoV-2 viral sequences, the mechanism also overcomes frequently observed limitations of LAMP assays arising from non-specific amplification within multiplexed reactions performed in a single \"pot\". Results showed excellent clinical performance (sensitivity 94.5%, specificity 100%, n = 190) when compared directly to a commercial gold standard reverse transcription quantitative polymerase chain reaction assay for the extracted RNA from nasopharyngeal samples and the capability of detecting a wide range of sequences containing at least alpha and delta variants. To further validate the test with no sample processing, directly from nasopharyngeal swabs, we also detected SARS-CoV-2 in positive clinical samples (n = 49), opening up the possibility for the assay's use in decentralized testing.","lamp_id":[{"id":"LPB00501","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00502","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"}]},{"id":263,"pmid":35081119,"title":"End-to-end system for rapid and sensitive early-detection of SARS-CoV-2 for resource-poor and field-test environments using a $51 lab-in-a-backpack","year":2022,"journal":"PLoS One","authors":"E Emily Lin, Umar A Razzaque, Stephen A Burrows, Stoyan K Smoukov","doi":"10.1371\/journal.pone.0259886","country":"United Kingdom","institute":"Queen Mary University of London","deparment":"School of Engineering and Materials Science","abstract":"COVID-19 has exposed stark inequalities between resource-rich and resource-poor countries. International UN- and WHO-led efforts, such as COVAX, have provided SARS-CoV-2 vaccines but half of African countries have less than 2% vaccinated in their population, and only 15 have reached 10% by October 2021, further disadvantaging local economic recovery. Key for this implementation and preventing further mutation and spread is the frequency of voluntary [asymptomatic] testing. It is limited by expensive PCR and LAMP tests, uncomfortable probes deep in the throat or nose, and the availability of hardware to administer in remote locations. There is an urgent need for an inexpensive \"end-to-end\" system to deliver sensitive and reliable, non-invasive tests in resource-poor and field-test conditions. We introduce a non-invasive saliva-based LAMP colorimetric test kit and a $51 lab-in-a-backpack system that detects as few as 4 viral RNA copies per \u03bcL. It consists of eight chemicals, a thermometer, a thermos bottle, two micropipettes and a 1000-4000 rcf electronically operated centrifuge made from recycled computer hard drives (CentriDrive). The centrifuge includes a 3D-printed rotor and a 12 V rechargeable Li-ion battery, and its 12 V standard also allows wiring directly to automobile batteries, to enable field-use of this and other tests in low infrastructure settings. The test takes 90 minutes to process 6 samples and has reagent costs of $3.5 per sample. The non-invasive nature of saliva testing would allow higher penetration of testing and wider adoption of the test across cultures and settings (including refugee camps and disaster zones). The attached graphical procedure would make the test suitable for self-testing at home, performing it in the field, or in mobile testing centers by minimally trained staff.","lamp_id":[{"id":"LPB00503","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":264,"pmid":34945432,"title":"Microfluidic Chip with Two-Stage Isothermal Amplification Method for Highly Sensitive Parallel Detection of SARS-CoV-2 and Measles Virus","year":2021,"journal":"Micromachines","authors":"Qin Huang, Xiaohui Shan, Ranran Cao, Xiangyu Jin, Xue Lin, Qiurong He, Yulei Zhu, Rongxin Fu, Wenli Du, Wenqi Lv, Ying Xia, Guoliang Huang","doi":"10.3390\/mi12121582","country":"China","institute":"Tsinghua University","deparment":"Department of Biomedical Engineering","abstract":"A two-stage isothermal amplification method, which consists of a first-stage basic recombinase polymerase amplification (RPA) and a second-stage fluorescence loop-mediated isothermal amplification (LAMP), as well as a microfluidic-chip-based portable system, were developed in this study; these enabled parallel detection of multiplex targets in real time in around one hour, with high sensitivity and specificity, without cross-contamination. The consumption of the sample and the reagent was 2.1 \u03bcL and 10.6 \u03bcL per reaction for RPA and LAMP, respectively. The lowest detection limit (LOD) was about 10 copies. The clinical amplification of about 40 nasopharyngeal swab samples, containing 17 SARS-CoV-2 (severe acute respiratory syndrome coronavirus) and 23 measles viruses (MV), were parallel tested by using the microfluidic chip. Both clinical specificity and sensitivity were 100% for MV, and the clinical specificity and sensitivity were 94.12% and 95.83% for SARS-CoV-2, respectively. This two-stage isothermal amplification method based on the microfluidic chip format offers a convenient, clinically parallel molecular diagnostic method, which can identify different nucleic acid samples simultaneously and in a timely manner, and with a low cost of the reaction reagent. It is especially suitable for resource-limited areas and point-of-care testing (POCT).","lamp_id":[{"id":"LPB00506","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00572","pathogen":"Measles morbillivirus","target":"P"}]},{"id":265,"pmid":35920555,"title":"SLIDE: Saliva-Based SARS-CoV-2 Self-Testing with RT-LAMP in a Mobile Device","year":2022,"journal":"ACS Sensors","authors":"Zifan Tang, Jiarui Cui, Aneesh Kshirsagar, Tianyi Liu, Michele Yon, Suresh V Kuchipudi, Weihua Guan","doi":"10.1021\/acssensors.2c01023","country":"USA","institute":"Pennsylvania State University","deparment":"Department of Electrical Engineering","abstract":"Regular, accurate, rapid, and inexpensive self-testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is urgently needed to quell pandemic propagation. The existing at-home nucleic acid testing (NAT) test has high sensitivity and specificity, but it requires users to mail the sample to the central lab, which often takes 3-5 days to obtain the results. On the other hand, rapid antigen tests for the SARS-CoV-2 antigen provide a fast sample to answer the test (15 min). However, the sensitivity of antigen tests is 30 to 40% lower than nucleic acid testing, which could miss a significant portion of infected patients. Here, we developed a fully integrated SARS-CoV-2 reverse transcription loop-mediated isothermal amplification (RT-LAMP) device using a self-collected saliva sample. This platform can automatically handle the complexity and can perform the functions, including (1) virus particles' thermal lysis preparation, (2) sample dispensing, (3) target sequence RT-LAMP amplification, (4) real-time detection, and (5) result report and communication. With a turnaround time of less than 45 min, our device achieved the limit of detection (LoD) of 5 copies\/\u03bcL of the saliva sample, which is comparable with the LoD (6 copies\/\u03bcL) using FDA-approved quantitative real-time polymerase chain reaction (qRT-PCR) assays with the same heat-lysis saliva sample preparation method. With clinical samples, our platform showed a good agreement with the results from the gold-standard RT-PCR method. These results show that our platform can perform self-administrated SARS-CoV-2 nucleic acid testing by laypersons with noninvasive saliva samples. We believe that our self-testing platform will have an ongoing benefit for COVID-19 control and fighting future pandemics.","lamp_id":[{"id":"LPB00508","pathogen":"SARS-CoV-2","target":"N"}]},{"id":266,"pmid":35982419,"title":"Clinical testing on SARS-CoV-2 swab samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP)","year":2022,"journal":"BMC Infectious Diseases","authors":"Meng Yee Lai, Fatma Diyana Mohd Bukhari, Nur Zulaikha Zulkefli, Ilyiana Ismail, Nur Izati Mustapa, Tuan Suhaila Tuan Soh, Afifah Haji Hassan, Kalaiarasu M Peariasamy, Yee Leng Lee, Jeyanthi Suppiah, Ravindran Thayan, Mohd Khairi Mat Isa, Nur Zafirah Abdul Wahid, Yee Ling Lau","doi":"10.1186\/s12879-022-07684-w","country":"Malaysia","institute":"Universiti Malaya","deparment":"Department of Parasitology","abstract":"Background: High cost of commercial RNA extraction kits limits the testing efficiency of SARS-CoV-2. Here, we developed a simple nucleic acid extraction method for the detection of SARS-CoV-2 directly from nasopharyngeal swab samples.\n\nMethods: A pH sensitive dye was used as the end point detection method. The obvious colour changes between positive and negative reactions eliminates the need of other equipment.\n\nResults: Clinical testing using 260 samples showed 92.7% sensitivity (95% CI 87.3-96.3%) and 93.6% specificity (95% CI 87.3-97.4%) of RT-LAMP.\n\nConclusions: The simple RNA extraction method minimizes the need for any extensive laboratory set-up. We suggest combining this simple nucleic acid extraction method and RT-LAMP technology as the point-of care diagnostic tool.","lamp_id":[{"id":"LPB00509","pathogen":"SARS-CoV-2","target":"N"}]},{"id":267,"pmid":35903482,"title":"A Visual Assay of a Loop-Mediated Isothermal Amplification Based Vertical Immunoassay for SARS-CoV-2 RNA Detection","year":2022,"journal":"Frontiers in Microbiology","authors":"Mengtao Yu, Pei Huang, Yuanguo Li, Yumeng Song, Xingqi Liu, Na Feng, Hongli Jin, Yujie Bai, Haili Zhang, Yuanyuan Li, Xianzhu Xia, Yuwei Gao, Hualei Wang","doi":"10.3389\/fmicb.2022.932698","country":"China","institute":"Jilin University","deparment":"State Key Laboratory for Zoonotic Diseases","abstract":"SARS-CoV-2 is a novel coronavirus that has caused a global pandemic. To date, 504,907,616 people have been infected and developed coronavirus disease 2019 (COVID-19). A rapid and simple diagnostic method is needed to control this pandemic. In this study, a visual nucleic acid detection method combining reverse transcription loop-mediated isothermal amplification and a vertical flow visualization strip (RT-LAMP-VF) was successfully established and could detect 20 copies\/\u03bcl of SARS-CoV-2 RNA transcript within 50 min at 61\u00b0C. This assay had no cross-reactivity with a variety of coronaviruses, including human coronavirus OC43, 229E, HKU1, NL63, severe acute respiratory syndrome-related coronavirus (SARSr-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and bat coronavirus HKU4, exhibiting very high levels of diagnostic sensitivity and specificity. Most strikingly, this method can be used for detecting multiple SARS-CoV-2 variants, including the Wuhan-Hu-1 strain, Delta, and Omicron variants. Compared with the RT-qPCR method recommended by the World Health Organization (WHO), RT-LAMP-VF does not require special equipment and is easy to perform. As a result, it is more suitable for rapid screening of suspected SARS-CoV-2 samples in the field and local laboratories.","lamp_id":[{"id":"LPB00510","pathogen":"SARS-CoV-2","target":"N"}]},{"id":268,"pmid":35620142,"title":"A new RT-LAMP-on-a-Chip Instrument for SARS-CoV-2 diagnostics","year":2022,"journal":"Microchemical Journal","authors":"Geovani Torezin Mendon\u00e7a, Mateus Cassaboni Stracke, Bruna de Oliveira Coelho, Heloisa Bruna Soligo Sanchuki, Viviane Klassen de Oliveira, Fabricio Klerynton Marchini, Dalila Luc\u00edola Zanette, Mateus N\u00f3brega Aoki, Emilson Ribeiro Viana , Lucas Blanes","doi":"10.1016\/j.microc.2022.107600","country":"Brazil","institute":"Carlos Chagas Institute","deparment":"Laboratory for Applied Science and Technology in Health","abstract":"This work describes the development of a Point-of-Care (POC) Lab-on-a-Chip (LOC) instrument for diagnosis of SARS-CoV-2 by Reverse-Transcription Loop-mediated isothermal amplification (RT-LAMP). The hardware is based on a Raspberry Pi computer ($35), a video camera, an Arduino Nano microcontroller, a printed circuit board as a heater and a 3D printed housing. The chips were manufactured in polymethyl methacrylate (PMMA) using a CO2 laser cutting machine and sealed with a PCR optic plastic film. The chip temperature is precisely controlled by a proportional-integral-derivative (PID) algorithm. During the RT-LAMP amplifications the chip was maintained at \u223c (65.0 \u00b1 0.1) \u00b0C for 25 minutes and 5 minutes cooling down, totaling a 30 minutes of reaction .The software interpretation occurs in less than a second. The chip design has four 25 \u00b5L chambers, two for clinical samples and two for positive and negative control-samples. The RT-LAMP master mix solution added in the chip chambers contains the pH indicator Phenol Red, that is pink (for pH \u223c 8.0) before amplification and becomes yellow (pH \u223c 6.0) if the genetic material is amplified. The RT-LAMP SARS-CoV-2 diagnostic was made by color image recognition using the OpenCV machine vision software library. The software was programmed to automatically distinguish the HSV color parameter distribution in each one of the four chip chambers. The instrument was successfully tested for SARS-CoV-2 diagnosis, in 22 clinic samples, 11 positives and 11 negatives, achieving an assertiveness of 86% when compared to the results obtained by RT-LAMP standard reactions performed in conventional PCR equipment.","lamp_id":[{"id":"LPB00511","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":269,"pmid":36048856,"title":"A rapid RT-LAMP SARS-CoV-2 screening assay for collapsing asymptomatic COVID-19 transmission","year":2022,"journal":"PLoS One","authors":"Rebecca C Allsopp, Caroline M Cowley, Ruth C Barber, Carolyn Jones, Christopher W Holmes, Paul W Bird, Shailesh G Gohil, Claire Blackmore, Martin D Tobin, Nigel Brunskill, Philip N Baker, Jacqui A Shaw","doi":"10.1371\/journal.pone.0273912","country":"United Kingdom","institute":"University of Leicester","deparment":"Department of Genetics and Genome Biology","abstract":"Purpose: To demonstrate the diagnostic performance of rapid SARS-CoV-2 RT-LAMP assays, comparing the performance of genomic versus sub-genomic sequence target with subsequent application in an asymptomatic screening population.\n\nMethods: RT-LAMP diagnostic specificity (DSe) and sensitivity (DSe) was determined using 114 RT-PCR clinically positive and 88 RT-PCR clinically negative swab samples processed through the diagnostic RT-PCR service within the University Hospitals of Leicester NHS Trust. A swab-based RT-LAMP SARS-CoV-2 screening programme was subsequently made available to all staff and students at the University of Leicester (Autumn 2020), implemented to ISO 15189:2012 standards using NHS IT infrastructure and supported by University Hospital Leicester via confirmatory NHS diagnostic laboratory testing of RT-LAMP 'positive' samples.\n\nResults: Validation samples reporting a Ct < 20 were detected at 100% DSe and DSp, reducing to 95% DSe (100% DSp) for all samples reporting a Ct < 30 (both genomic dual sub-genomic assays). Advisory screening identified nine positive cases in 1680 symptom free individuals (equivalent to 540 cases per 100,000) with results reported back to participants and feed into national statistics within 48 hours.\n\nConclusion: This work demonstrates the utility of a rapid RT-LAMP assay for collapsing transmission of SARS-CoV-2 in an asymptomatic screening population.","lamp_id":[{"id":"LPB00512","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00513","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00514","pathogen":"SARS-CoV-2","target":"N"}]},{"id":270,"pmid":33024053,"title":"End-to-End Protocol for the Detection of SARS-CoV-2 from Built Environments","year":2020,"journal":"mSystems","authors":"Ceth W. Parker, Nitin Singh, Scott Tighe, Adriana Blachowicz, Jason M. Wood, Arman Seuylemezian, Parag Vaishampayan, Camilla Urbaniak, Ryan Hendrickson, Pheobe Laaguiby, Kevin Clark, Brian G. Clement, Niamh B. O\u2019Hara, Mara Couto-Rodriguez, Daniela Bezdan, Christopher E. Mason, Kasthuri Venkateswaran","doi":"10.1128\/mSystems.00771-20","country":"","institute":"","deparment":"","abstract":"Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is a respiratory virus primarily transmitted person to person through inhalation of droplets or aerosols, laden with viral particles. However, as recent studies have shown, virions can remain infectious for up to 72\u2009h on surfaces, which can lead to transmission through contact. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end (E2E) study showed that the effective combination for monitoring SARS-CoV-2 on surfaces includes using an Isohelix swab collection tool, DNA\/RNA Shield as a preservative, an automated system for RNA extraction, and reverse transcriptase quantitative PCR (RT-qPCR) as the detection assay. Using this E2E approach, this study showed that, in some cases, noninfectious viral fragments of SARS-CoV-2 persisted on surfaces for as long as 8 days even after bleach treatment. Additionally, debris associated with specific built environment surfaces appeared to inhibit and negatively impact the recovery of RNA; Amerstat demonstrated the highest inhibition (>90%) when challenged with an inactivated viral control. Overall, it was determined that this E2E protocol required a minimum of 1,000 viral particles per 25 cm^2 to successfully detect virus from test surfaces. Despite our findings of viral fragment longevity on surfaces, when this method was employed to evaluate 368 samples collected from various built environmental surfaces, all samples tested negative, indicating that the surfaces were either void of virus or below the detection limit of the assay.","lamp_id":[{"id":"LPB00515","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00714","pathogen":"SARS-CoV-2","target":"N"}]},{"id":271,"pmid":35798777,"title":"Detecting lineage-defining mutations in SARS-CoV-2 using colorimetric RT-LAMP without probes or additional primers","year":2022,"journal":"Scientific Reports","authors":"Carlos Abelardo Dos Santos, L\u00edvia do Carmo Silva, Marcio Neres de Souza J\u00fanior, Geovana de Melo Mendes, Paulo Felipe Neves Estrela, K\u00e9zia Gomes de Oliveira, Juliana Santana de Curcio, Paola Cristina Resende, Marilda Mendon\u00e7a Siqueira, Alex Pauvolid-Corr\u00eaa, Gabriela Rodrigues Mendes Duarte, Elis\u00e2ngela de Paula Silveira-Lacerda","doi":"10.1038\/s41598-022-15368-3","country":"Brazil","institute":"Instituto de Ci\u00eancias Biol\u00f3gicas I","deparment":"Departamento de Gen\u00e9tica","abstract":"Despite the advance of vaccination worldwide, epidemic waves caused by more transmissible and immune evasive genetic variants of SARS-CoV-2 have sustained the ongoing pandemic of COVID-19. Monitoring such variants is expensive, as it usually relies on whole-genome sequencing methods. Therefore, it is necessary to develop alternatives that could help identify samples from specific variants. Reverse transcription loop-mediated isothermal amplification is a method that has been increasingly used for nucleic acid amplification, as it is cheaper and easier to perform when compared to other molecular techniques. As a proof of concept that can help distinguish variants, we present an RT-LAMP assay capable of detecting samples carrying a group of mutations that can be related to specific SARS-CoV-2 lineages, here demonstrated for the Variant of Concern Gamma. We tested 60 SARS-CoV-2 RNA samples extracted from swab samples and the reaction showed a sensitivity of 93.33%, a specificity of 88.89% and a kappa value of 0.822 for samples with a Ct \u2264 22.93. The RT-LAMP assay demonstrated to be useful to distinguish VOC Gamma and may be of particular interest as a screening approach for variants in countries with poor sequencing coverage.","lamp_id":[{"id":"LPB00516","pathogen":"SARS-CoV-2 (Gamma)","target":"S"}]},{"id":272,"pmid":35797288,"title":"Development of a colorimetric RT-LAMP assay for the detection of SARS-COV-2 isolated from Oman","year":2022,"journal":"Journal of Infection in Developing Countries","authors":"Haytham Ali, Khaddia Alkhaursi, Timothy Holton","doi":"10.3855\/jidc.15377","country":"Oman","institute":"Sultan Qaboos University","deparment":"Department of Animal and Veterinary Sciences","abstract":"Introduction: A rapid and sensitive COVID-19 diagnostic test is required to aid in the prevention and control of the current COVID-19 pandemic spread. We developed a colorimetric, rapid, and sensitive RT-LAMP assay for the diagnosis of COVID-19 viral infection.\n\nMethodology: Complete genome sequences of 41 SARS-CoV-2 isolates from Oman were used in this study. Three primer sets (CoV_S1, CoV_S2, CoV_M1) were developed from all Omani SARS-CoV-2 genome sequences available at the time, targeting the spike protein gene and the M gene. The primer set (CoV_S1) was found to be the most sensitive and specific among the three designed sets. The sensitivity and specificity of the assay were compared to that of qRT-PCR. Direct testing of SARS-CoV-2 spiked saliva with the developed assay was evaluated. Lyophilized colorimetric assays were stored at room temperature and 4 \u00b0C and their ability to detect positive samples were tested for a period of 8 weeks.\n\nResults: The RT-LAMP assay was validated by testing 145 COVID-19 clinical samples with a sensitivity of 96.9% and specificity of 94.7% when compared to the validated qRT-PCR assay. The assay specificity was tested against SARS-CoV Frankfurt 1 RNA virus and avian coronaviruses as they tested negative with the developed assay. The assay was lyophilized and managed to detect the positive samples colorimetrically when stored at 4 \u00b0C for up to 8 weeks.\n\nConclusions: The assay can be utilized in its current form as a screening assay with the advantages of being simpler, quicker, and cheaper than the qRT-PCR.","lamp_id":[{"id":"LPB00517","pathogen":"SARS-CoV-2","target":"N"}]},{"id":273,"pmid":35540180,"title":"Diagnostic utility and validation of a newly developed real time loop mediated isothermal amplification method for the detection of SARS CoV-2 infection","year":2022,"journal":"Journal of Clinical Virology Plus","authors":"Bushran N Iqbal, Shiyamalee Arunasalam, Maduja V M Divarathna, Aaom Jabeer, Pdnn Sirisena, Thamarasi Senaratne, Rohitha Muthugala, Faseeha Noordeen","doi":"10.1016\/j.jcvp.2022.100081","country":"Sri Lanka","institute":"University of Peradeniya","deparment":"Department of Microbiology","abstract":"Background: Detecting SARS-CoV-2 using a simple real time molecular assay will be helpful for the mitigation efforts in low \/ middle income countries during the pandemic. We have developed and validated a rapid and simple real time loop mediated isothermal amplification assay (LAMP) for screening of SARS-CoV-2 infection in known infected and non-infected individuals.\n\nMethods: Six sets of primers were designed targeting the N-gene of the SARS-CoV-2 (Accession ID MN994468). LAMP reactions were performed using Warm Start 2X Master Mix and real-time PCR machine at 65 \u00b0C for 60 cycles with 15 s for each cycle. Results were read by visualizing turbidity under ultraviolet light and real time fluorescence detection through FAM channel of the real time PCR machine. We tested a total of 320 including 240 SARS CoV-2 positive (Ct values <40) and 80 SARS CoV-2 negative samples as tested by a real time RT-PCR using the newly developed LAMP assay.\n\nResults: A total of 206 out of 240 SARS CoV-2 positive samples were tested positive by the newly developed LAMP assay with a sensitivity of 86%. All 80 SARS CoV-2 negative samples were tested negative by the newly developed LAMP assay with a specificity of 100%.\n\nConclusion: The newly developed real time LAMP assay has a sensitivity of 86% and specificity of 100% compared to the real time RT-PCR for the detection of SARS CoV-2. The new assay will be useful to screen large number of samples if adopted to minimize the time and cost.","lamp_id":[{"id":"LPB00518","pathogen":"SARS-CoV-2","target":"N"}]},{"id":274,"pmid":35535440,"title":"Detection of SARS-CoV-2 in different human biofluids using the loop-mediated isothermal amplification assay: A prospective diagnostic study in Fortaleza, Brazil","year":2022,"journal":"Journal of Medical Virology","authors":"Marco Clementino, Karene F Cavalcante, Vania A F Viana, Dayara de Oliveira Silva, Caroline R Damasceno, Jessica Fernandes de Souza, Rafhaella N D G Gondim, Daniel M de Melo Jorge, Lyvia M V C Magalh\u00e3es, \u00c9rico A G de Arruda, Roberto da J P Neto, Melissa S Medeiros, Arm\u00eanio A Dos Santos, Pedro J C Magalh\u00e3es, Liana P Mello, Eurico Arruda, Aldo \u00c2 M Lima, Alexandre Havt","doi":"10.1002\/jmv.27842","country":"Brazil","institute":"Universidade Federal do Cear\u00e1","deparment":"INCT-Biomedicina no Semi\u00e1rido Brasileiro","abstract":"We adopted the reverse-transcriptase-loop-mediated isothermal amplification (RT-LAMP) to detect severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) in patient samples. Two primer sets for genes N and Orf1ab were designed to detect SARS-CoV-2, and one primer set was designed to detect the human gene Actin. We collected prospective 138 nasopharyngeal swabs, 70 oropharyngeal swabs, 69 salivae, and 68 mouth saline wash samples from patients suspected to have severe acute respiratory syndrome (SARS) caused by SARS-CoV-2 to test the RT-LAMP in comparison with the gold standard technique reverse-transcription quantitative polymerase chain reaction (RT-qPCR). The accuracy of diagnosis using both primers, N5 and Orf9, was evaluated. Sensitivity and specificity for diagnosis were 96% (95% confidence interval [CI]: 87-99) and 85% (95% CI: 76-91) in 138 samples, respectively. Accurate diagnosis results were obtained only in nasopharyngeal swabs processed via extraction kit. Accurate and rapid diagnosis could aid coronavirus disease 2019 (COVID-19) pandemic management by identifying, isolating, and treating patients rapidly.","lamp_id":[{"id":"LPB00519","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00520","pathogen":"SARS-CoV-2","target":"N"}]},{"id":275,"pmid":35521217,"title":"Elimination of Carryover Contamination in Real-Time Reverse Transcriptase Loop-Mediated Isothermal Amplification for Rapid Detection of the SARS-CoV-2 Virus in Point-of-Care Testing","year":2022,"journal":"Frontiers in Cellular and Infection Microbiology","authors":"Than Linh Quyen, Aaydha Chidambara Vinayaka, Mohsen Golabi, Huynh Van Ngoc, Dang Duong Bang, Anders Wolff","doi":"10.3389\/fcimb.2022.856553","country":"Denmark","institute":"Technical University of Denmark","deparment":"Department of Bioengineering","abstract":"Loop-mediated isothermal amplification (LAMP) is being used as a robust rapid diagnostic tool to prevent the transmission of infectious diseases. However, carryover contamination of LAMP-amplified products originating from previous tests has been a problem in LAMP-based bio-analytical assays. In this study, we developed a Cod-uracil-DNA-glycosylase real-time reverse transcriptase LAMP assay (Cod-UNG-rRT-LAMP) for the elimination of carryover contamination and the rapid detection of SARS-CoV-2 in point-of-care (POC) testing. Using the Cod-UNG-rRT-LAMP assay, the SARS-CoV-2 virus could be detected as low as 2 copies\/\u00b5l (8 copies\/reaction) within 45 min of amplification and 2.63 \u00b1 0.17 pg (equivalent to 2.296 \u00d7 10^9 copies) of contaminants per reaction could be eliminated. Analysis of clinical SARS-CoV-2 samples using the Cod-UNG-rRT-LAMP assay showed an excellent agreement with a relative accuracy of 98.2%, sensitivity of 97.1%, and specificity of 95.2% in comparison to rRT-PCR. The results obtained in this study clearly demonstrate the feasibility of the use of the Cod-UNG-rRT-LAMP assay for applications toward the POC diagnosis of SARS-CoV-2 and on-site testing of other pathogens.","lamp_id":[{"id":"LPB00521","pathogen":"SARS-CoV-2","target":"N"}]},{"id":276,"pmid":35670202,"title":"Automated sample-to-answer centrifugal microfluidic system for rapid molecular diagnostics of SARS-CoV-2","year":2022,"journal":"Lab on a Chip","authors":"Lidija Malic, Daniel Brassard, Dillon Da Fonte, Christina Nassif, Maxence Mounier, Andr\u00e9 Ponton, Matthias Geissler, Matthew Shiu, Keith J Morton, Teodor Veres","doi":"10.1039\/d2lc00242f","country":"Canada","institute":"National Research Council of Canada","deparment":"Life Sciences Division","abstract":"Testing for SARS-CoV-2 is one of the most important assets in COVID-19 management and mitigation. At the onset of the pandemic, SARS-CoV-2 testing was uniquely performed in central laboratories using RT-qPCR. RT-qPCR relies on trained personnel operating complex instrumentation, while time-to-result can be lengthy (e.g., 24 to 72 h). Now, two years into the pandemic, with the surge in cases driven by the highly transmissible Omicron variant, COVID-19 testing capabilities have been stretched to their limit worldwide. Rapid antigen tests are playing an increasingly important role in quelling outbreaks by expanding testing capacity outside the realm of clinical laboratories. These tests can be deployed in settings where repeat and rapid testing is essential, but they often come at the expense of limited accuracy and sensitivity. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) provides a number of advantages to SARS-CoV-2 testing in standard laboratories and at the point-of-need. In contrast to RT-qPCR, RT-LAMP is performed at a constant temperature, which circumvents the need for thermal cycling and translates into a shorter analysis time (e.g., <1 h). In addition, RT-LAMP is compatible with colorimetric detection, facilitating visualization and read-out. However, even with these benefits, RT-LAMP is not yet clinically deployed at its full capacity. Lack of automation and integration of sample preparation, such as RNA extraction, limits the sensitivity and specificity of the method. Furthermore, the need for cold storage of reagents complicates its use at the point of need. The developments presented in this work address these limitations: We describe a fully automated SARS-CoV-2 detection method using RT-LAMP, which also includes up-front lysis and extraction of viral RNA, performed on a centrifugal platform with active pneumatic pumping, a disposable, all-polymer-based microfluidic cartridge and lyophilized reagents. We demonstrate that the limit of detection of the RT-LAMP assay itself is 0.2 copies per \u03bcL using N and E genes as target sequences. When combined with integrated RNA extraction, the assay sensitivity is 0.5 copies per \u03bcL, which is highly competitive to RT-qPCR. We tested the automated assay using 12 clinical swab specimens from patients and were able to distinguish positive and negative samples for SARS-CoV-2 within 60 min, thereby obtaining 100% agreement with RT-qPCR results.","lamp_id":[{"id":"LPB00522","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00523","pathogen":"SARS-CoV-2","target":"N"}]},{"id":277,"pmid":35501862,"title":"A fast extraction-free isothermal LAMP assay for detection of SARS-CoV-2 with potential use in resource-limited settings","year":2022,"journal":"Virology Journal","authors":"Kathleen G\u00e4rtner, Harry Meleke, Mercy Kamdolozi, David Chaima, Lyson Samikwa, Mary Paynter, Maggie Nyirenda Nyang'Wa, Elaine Cloutman-Green, Eleni Nastouli, Nigel Klein, Tonney Nyirenda, Chisomo Msefula, Dagmar G Alber","doi":"10.1186\/s12985-022-01800-7","country":"United Kingdom","institute":"University College London","deparment":"Great Ormond Street Institute of Child Health","abstract":"Background: To retain the spread of SARS-CoV-2, fast, sensitive and cost-effective testing is essential, particularly in resource limited settings (RLS). Current standard nucleic acid-based RT-PCR assays, although highly sensitive and specific, require transportation of samples to specialised laboratories, trained staff and expensive reagents. The latter are often not readily available in low- and middle-income countries and this may significantly impact on the successful disease management in these settings. Various studies have suggested a SARS-CoV-2 loop mediated isothermal amplification (LAMP) assay as an alternative method to RT-PCR.\n\nMethods: Four previously published primer pairs were used for detection of SARS-CoV-2 in the LAMP assay. To determine optimal conditions, different temperatures, sample input and incubation times were tested. Ninety-three extracted RNA samples from St. George's Hospital, London, 10 non-extracted nasopharyngeal swab samples from Great Ormond Street Hospital for Children, London, and 92 non-extracted samples from Queen Elisabeth Central Hospital (QECH), Malawi, which have previously been tested for SARS-Cov-2 by quantitative reverse-transcription RealTime PCR (qRT-PCR), were analysed in the LAMP assay.\n\nResults: In this study we report the optimisation of an extraction-free colourimetric SARS-CoV-2 LAMP assay and demonstrated that a lower limit of detection (LOD) between 10 and 100 copies\/\u00b5L of SARS-CoV-2 could be readily detected by a colour change of the reaction within as little as 30 min. We further show that this assay could be quickly established in Malawi, as no expensive equipment is necessary. We tested 92 clinical samples from QECH and showed the sensitivity and specificity of the assay to be 86.7% and 98.4%, respectively. Some viral transport media, used routinely to stabilise RNA in clinical samples during transportation, caused a non-specific colour-change in the LAMP reaction and therefore we suggest collecting samples in phosphate buffered saline (which did not affect the colour) as the assay allows immediate sample analysis on-site.\n\nConclusion: SARS-CoV-2 LAMP is a cheap and reliable assay that can be readily employed in RLS to improve disease monitoring and management.","lamp_id":[{"id":"LPB00524","pathogen":"SARS-CoV-2","target":"N"}]},{"id":278,"pmid":35501506,"title":"Rapid and simple detection of influenza virus via isothermal amplification lateral flow assay","year":2022,"journal":"Analytical and Bioanalytical Chemistry","authors":"Minju Jang, SeJin Kim, Junkyu Song, Sanghyo Kim","doi":"10.1007\/s00216-022-04090-8","country":"Republic of Korea","institute":"Gachon University","deparment":"Department of Bionanotechnology","abstract":"Respiratory illness caused by influenza virus is a serious public health problem worldwide. As the symptoms of influenza virus infection are similar to those of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, it is essential to distinguish these two viruses. Therefore, to properly respond to a pathogen, a detection method that is capable of rapid and accurate diagnosis in a hospital or at home is required. To satisfy this need, we applied loop-mediated isothermal amplification (LAMP) and an isothermal nucleic acid amplification technique, along with a system to analyze the results without specialized equipment, a lateral flow assay (LFA). Using the platform developed in this study, all processes, from sample preparation to detection, can be performed without special equipment. Unlike existing PCR methods, the nucleic acid amplification can be performed in the field because hot packs do not require electricity. Thus, the designed platform can provide rapid results without the need to transport the samples to a laboratory or hospital. These advantages are not limited to operations in developing countries with poor access to medical systems. In conclusion, the developed technology is a promising tool for infectious disease management that allows for rapid identification of infectious diseases and appropriate treatment of patients.","lamp_id":[{"id":"LPB00525","pathogen":"Influenza A virus (H1N1)","target":"M1"},{"id":"LPB00526","pathogen":"Influenza B virus","target":"NS1"}]},{"id":279,"pmid":35404944,"title":"A rapid, specific, extraction-less, and cost-effective RT-LAMP test for the detection of SARS-CoV-2 in clinical specimens","year":2022,"journal":"PLoS One","authors":"Francesco Elia Marino, Eric Proffitt, Eugene Joseph, Arun Manoharan","doi":"10.1371\/journal.pone.0266703","country":"USA","institute":"Prime Discoveries INC","deparment":"Department of Research and Development","abstract":"In 2019 a newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread rapidly from the epicenter in Wuhan (China) to more than 150 countries around the world, causing the Coronavirus disease 2019 (COVID-19) pandemic. In this study, we describe an extraction-less method based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) intended for the rapid qualitative detection of nucleic acid from SARS-CoV-2 in upper respiratory specimens, including oropharyngeal and nasopharyngeal swabs, anterior nasal and mid-turbinate nasal swabs, nasopharyngeal washes\/aspirates or nasal aspirates as well as bronchoalveolar lavage (BAL) from individuals suspected of COVID-19 by their healthcare provider. The assay's performance was evaluated and compared to an RT quantitative PCR-based assay (FDA-approved). With high sensitivity, specificity, and bypassing the need for RNA extraction, the RT-LAMP Rapid Detection assay is a valuable and fast test for an accurate and rapid RNA detection of the SARS-CoV-2 virus and potentially other pathogens. Additionally, the versatility of this test allows its application in virtually every laboratory setting and remote location where access to expensive laboratory equipment is a limiting factor for testing during pandemic crises.","lamp_id":[{"id":"LPB00527","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00528","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00529","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":280,"pmid":35453876,"title":"Rapid and Reliable Detection of SARS-CoV-2 Using Direct RT-LAMP","year":2022,"journal":"Diagnostics","authors":"Sherif A El-Kafrawy, Mai M El-Daly, Ahmed M Hassan, Steve M Harakeh, Thamir A Alandijany, Esam I Azhar","doi":"10.3390\/diagnostics12040828","country":"Saudi Arabia","institute":"King Abdulaziz University","deparment":"Special Infectious Agents Unit-BSL3, King Fahd Medical Research Center","abstract":"Background: The global pandemic coronavirus SARS-CoV-2 has a healthcare, social and economic burden. To limit the spread of the virus, the World Health Organization (WHO) urgently called for extensive screening of suspected individuals; thus, a quick, simple, and sensitive diagnostic assay is always in need. Methods: We applied reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for the detection of SARS-CoV-2. The RT-LAMP method was optimized by evaluating two fluorescence amplification mixes and several reaction times, and results were compared to the standard real-time RT-PCR (rtRT-PCR). The assay was validated using 200 nasopharyngeal swabs collected in viral transport media (62 positive for SARS-CoV-2, and 138 negative for SARS-CoV-2 detected by the rtRT-PCR method). The samples were diluted 1:4 in diethylpyrocarbonate (DEPC)-treated water, utilized for RT-LAMP using different singleplex and multiplex sets of LAMP primers (N gene, S gene, and orf1ab gene), and incubated at 65 \u00b0C using real-time PCR 7500. Results: Our direct detection with the RT-LAMP protocol showed 100% concordance (sensitivity and specificity) with the standard protocol used for the detection of SARS-CoV-2 nucleic acid. Conclusions: In this study, we set up a rapid, simple, and sensitive RT-LAMP assay for the detection of SARS-CoV-2 in clinical samples. The assay is suitable for point of care detection in public hospitals, medical centers in rural areas, and in transportation hubs.","lamp_id":[{"id":"LPB00530","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00536","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00553","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":281,"pmid":35381892,"title":"Dual-site ligation-assisted loop-mediated isothermal amplification (dLig-LAMP) for colorimetric and point-of-care determination of real SARS-CoV-2","year":2022,"journal":"Microchimica Acta","authors":"Moon Hyeok Choi, Jaehyeon Lee, Young Jun Seo","doi":"10.1007\/s00604-022-05293-7","country":"South Korea","institute":"Jeonbuk National University","deparment":"Department of Chemistry","abstract":"A probing system has been developed based on dual-site ligation-assisted loop-mediated isothermal amplification (dLig-LAMP) for the selective colorimetric detection of SARS-CoV-2. This approach can induce false-positive and -negative detection in real clinical samples; dLig-LAMP operates with improved selectivity. Unlike RT-LAMP, the selectivity of dLig-LAMP is determined in both the ligation and primer binding steps, not in the reverse transcription step. With this selective system in hand, we developed a colorimetric signaling system for point-of-care detection. We also developed a colorimetric probe for sensing pyrophosphate, which arises as a side product during the LAMP DNA amplification. Thus, dLig-LAMP appears to be an alternative method for improving the selectivity problems associated with reverse transcription. In addition, combining dLig-LAMP with colorimetric pyrophosphate probing allows point-of-care detection of SARS-CoV-2 within 1 h with high selectivity.","lamp_id":[{"id":"LPB00531","pathogen":"SARS-CoV-2","target":"N"}]},{"id":282,"pmid":35766492,"title":"COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme","year":2022,"journal":"Journal of Clinical Microbiology","authors":"Clare L Fasching, Venice Servellita, Bridget McKay, Vaishnavi Nagesh, James P Broughton, Alicia Sotomayor-Gonzalez, Baolin Wang, Noah Brazer, Kevin Reyes, Jessica Streithorst, Rachel N Deraney, Emma Stanfield, Carley G Hendriks, Becky Fung, Steve Miller, Jesus Ching, Janice S Chen, Charles Y Chiu","doi":"10.1128\/jcm.00261-22","country":"USA","institute":"Mammoth Biosciences","deparment":"","abstract":"Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here, we present the development and validation of a rapid COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K\/Q\/A, and N501Y mutations, at least one of which is found in nearly all major variants. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all targeted SNP mutations. An analysis pipeline for CRISPR-based SNP identification from 261 clinical samples yielded a SNP concordance of 97.3% and agreement of 98.9% (258 of 261) for SARS-CoV-2 lineage classification, using SARS-CoV-2 whole-genome sequencing and\/or real-time RT-PCR as test comparators. We also showed that detection of the single E484A mutation was necessary and sufficient to accurately identify Omicron from other major circulating variants in patient samples. These findings demonstrate the utility of CRISPR-based DETECTR as a faster and simpler diagnostic method compared with sequencing for SARS-CoV-2 variant identification in clinical and public health laboratories.","lamp_id":[{"id":"LPB00532","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00533","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00534","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00535","pathogen":"SARS-CoV-2","target":"S"}]},{"id":283,"pmid":35735410,"title":"Smartphone-Based SARS-CoV-2 and Variants Detection System using Colorimetric DNAzyme Reaction Triggered by Loop-Mediated Isothermal Amplification (LAMP) with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)","year":2022,"journal":"ACS Nano","authors":"Jayeon Song, Baekdong Cha, Jeong Moon, Hyowon Jang, Sunjoo Kim, Jieun Jang, Dongeun Yong, Hyung-Jun Kwon, In-Chul Lee, Eun-Kyung Lim, Juyeon Jung, Hyun Gyu Park, Taejoon Kang","doi":"10.1021\/acsnano.2c04840","country":"Republic of Korea","institute":"Korea Research Institute of Bioscience and Biotechnology (KRIBB)","deparment":"Bionanotechnology Research Center","abstract":"Coronavirus disease (COVID-19) has affected people for over two years. Moreover, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has raised concerns regarding its accurate diagnosis. Here, we report a colorimetric DNAzyme reaction triggered by loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR), referred to as DAMPR assay for detecting SARS-CoV-2 and variants genes with attomolar sensitivity within an hour. The CRISPR-associated protein 9 (Cas9) system eliminated false-positive signals of LAMP products, improving the accuracy of DAMPR assay. Further, we fabricated a portable DAMPR assay system using a three-dimensional printing technique and developed a machine learning (ML)-based smartphone application to routinely check diagnostic results of SARS-CoV-2 and variants. Among blind tests of 136 clinical samples, the proposed system successfully diagnosed COVID-19 patients with a clinical sensitivity and specificity of 100% each. More importantly, the D614G (variant-common), T478K (delta-specific), and A67V (omicron-specific) mutations of the SARS-CoV-2 S gene were detected selectively, enabling the diagnosis of 70 SARS-CoV-2 delta or omicron variant patients. The DAMPR assay system is expected to be employed for on-site, rapid, accurate detection of SARS-CoV-2 and its variants gene and employed in the diagnosis of various infectious diseases.","lamp_id":[{"id":"LPB00537","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00538","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00539","pathogen":"SARS-CoV-2","target":"S"}]},{"id":284,"pmid":35696983,"title":"Parallel multistep digital analysis SlipChip demonstrated with the quantification of nucleic acid by digital LAMP-CRISPR","year":2022,"journal":"Lab on a Chip","authors":"Ziqing Yu, Lei Xu, Weiyuan Lyu, Feng Shen","doi":"10.1039\/d2lc00284a","country":"China","institute":"Shanghai Jiao Tong University","deparment":"School of Biomedical Engineering","abstract":"Digital biological analysis compartmentalizes targets of interest, such as nucleic acids, proteins, and cells, to a single event level and performs detection and further investigation. Microfluidic-based digital biological analysis methods, including digital PCR, digital protein analysis, and digital cell analysis, have demonstrated superior advantages in research applications and clinical diagnostics. However, most of the methods are still based on a one-step \"divide and detect\" strategy, and it is challenging for these methods to perform further parallel manipulation of reaction partitions to achieve \"divide, manipulate, and analyze\" capabilities. Here, we present a parallel multistep digital analysis (PAMDA) SlipChip for the parallel multistep manipulation of a large number of droplets for digital biological analysis, demonstrated by the quantification of SARS-CoV-2 nucleic acids by a two-step digital isothermal amplification combined with clustered regularly interspaced short palindromic repeats (CRISPR). This PAMDA SlipChip utilizes a \"chain-of-pearl\" channel with a self-partitioning droplet formation mechanism that does not require the precise alignment of microfeatures for fluidic loading as the traditional SlipChip design. This device can first generate 2400 3.2 nanoliter droplets to perform digital loop-mediated isothermal amplification (LAMP) and then deliver reagents containing Cas12a protein and crRNA to each individual partition in parallel to simultaneously initiate digital CRISPR detection by a simple multistep slipping operation. This PAMDA SlipChip not only provides a promising tool to perform digital CRISPR with a flexible assay and workflow design but can also be applied for a broad range of applications in digital biological analysis that require multistep manipulation of partitions in parallel.","lamp_id":[{"id":"LPB00540","pathogen":"SARS-CoV-2","target":"N"}]},{"id":285,"pmid":34683202,"title":"A Portable Device for LAMP Based Detection of SARS-CoV-2","year":2021,"journal":"Micromachines","authors":"Kamalalayam Rajan Sreejith, Muhammad Umer, Larissa Dirr, Benjamin Bailly, Patrice Guillon, Mark von Itzstein, Narshone Soda, Surasak Kasetsirikul, Muhammad J. A. Shiddiky, Nam-Trung Nguyen","doi":"10.3390\/mi12101151","country":"","institute":"","deparment":"","abstract":"This paper reports the design, development, and testing of a novel, yet simple and low-cost portable device for the rapid detection of SARS-CoV-2. The device performs loop mediated isothermal amplification (LAMP) and provides visually distinguishable images of the fluorescence emitted from the samples. The device utilises an aluminium block embedded with a cartridge heater for isothermal heating of the sample and a single-board computer and camera for fluorescence detection. The device demonstrates promising results within 20 min using clinically relevant starting concentrations of the synthetic template. Time-to-signal data for this device are considerably lower compared to standard quantitative Polymerase Chain Reaction(qPCR) machine (~10\u201320 min vs. >38 min) for 1 \u00d7 10^2 starting template copy number. The device in its fully optimized and characterized state can potentially be used as simple to operate, rapid, sensitive, and inexpensive platform for population screening as well as point-of-need severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) detection and patient management.","lamp_id":[{"id":"LPB00541","pathogen":"SARS-CoV-2","target":"RdRp"}]},{"id":286,"pmid":35603267,"title":"Clinical validation of engineered CRISPR\/Cas12a for rapid SARS-CoV-2 detection","year":2022,"journal":"Communications Medicin","authors":"Long T Nguyen, Santosh R Rananaware, Brianna L M Pizzano, Brandon T Stone, Piyush K Jain","doi":"10.1038\/s43856-021-00066-4","country":"USA","institute":"University of Florida","deparment":"Department of Chemical Engineering","abstract":"Background: The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies have emerged as a rapid and affordable platform that can shape the future of diagnostics.\n\nMethods: We developed ENHANCEv2 that is composed of a chimeric guide RNA, a modified LbCas12a enzyme, and a dual reporter construct to improve the previously reported ENHANCE system. We validated both ENHANCE and ENHANCEv2 using 62 nasopharyngeal swabs and compared the results to RT-qPCR. We created a lyophilized version of ENHANCEv2 and characterized its detection capability and stability.\n\nResults: Here we demonstrate that when coupled with an RT-LAMP step, ENHANCE detects COVID-19 samples down to a few copies with 95% accuracy while maintaining a high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. ENHANCE works robustly in a wide range of magnesium concentrations (3 mM-13 mM), allowing for further assay optimization. Our clinical validation results for both ENHANCE and ENHANCEv2 show 60\/62 (96.7%) sample agreement with RT-qPCR results while only using 5 \u00b5L of sample and 20 minutes of CRISPR reaction. We show that the lateral flow assay using paper-based strips displays 100% agreement with the fluorescence-based reporter assay during clinical validation. Finally, we demonstrate that a lyophilized version of ENHANCEv2 shows high sensitivity and specificity for SARS-CoV-2 detection while reducing the CRISPR reaction time to as low as 3 minutes while maintaining its detection capability for several weeks upon storage at room temperature.\n\nConclusions: CRISPR-based diagnostic platforms offer many advantages as compared to conventional qPCR-based detection methods. Our work here provides clinical validation of ENHANCE and its improved form ENHANCEv2 for the detection of COVID-19.","lamp_id":[{"id":"LPB00542","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00543","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00544","pathogen":"SARS-CoV-2","target":"E"}]},{"id":287,"pmid":35284091,"title":"T-Cup: A Cheap, Rapid, and Simple Home Device for Isothermal Nucleic Acid Amplification","year":2021,"journal":"Global Challenges","authors":"Aldrik H Velders, Michel Ossendrijver, Bart J F Keijser, Vittorio Saggiomo","doi":"10.1002\/gch2.202100078","country":"The Netherlands","institute":"Wageningen University","deparment":"Laboratory of BioNanoTechnology","abstract":"During the SARS-CoV2 pandemic, it has become clear that centralized testing suffers from multiple bottlenecks. Logistics, number of machines, and people available to run the diagnostic tests are limited. A solution to those bottlenecks would be a fully decentralized system, where people can test themselves at home and only report back the outcome of the test in a centralized database. Here a noninstrumental device capable of achieving isothermal conditions useful for detecting the SARS-CoV2 RNA using loop mediated amplification (LAMP) tests is presented. This device, compared to others reported in literature or present on the market, is cheap, easy to produce and use, and has little impact on the environment. Using a simple aluminum coffee capsule, a phase change material, and a 3D printed holder, this device, when placed in boiling water, is able to maintain a temperature of 65 \u00b0C for 25 min, required for running the LAMP reaction. In principle, this device can be applied to any LAMP reaction, and hence employed for many different applications, and can be deployed in large quantities in short amount of time.","lamp_id":[{"id":"LPB00545","pathogen":"SARS-CoV-2","target":"E"}]},{"id":288,"pmid":35313427,"title":"Development of a RT-LAMP assay for detection of SARS-CoV-2","year":2022,"journal":"Indian Journal of Medical Research","authors":"Shyam Sundar Nandi, Upendra Pradeep Lambe, Sonali Ankush Sawant, Trupti Gohil, Jagadish Deshpande","doi":"10.4103\/ijmr.IJMR_713_21","country":"India","institute":"Haffkine Institute Compound","deparment":"ICMR-National Institute of Virology","abstract":"Background & objectives: The pandemic of SARS-COV-2 began in Wuhan, China in December 2019 and has caused more than 101 million cases worldwide. Diagnostic technologies possessing sensitivity and specificity equivalent to real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assays are needed to ramp up testing capacity in most countries. Newer platforms need to be technically less demanding, require minimum equipment and reduce turn-around time for reporting results. The objective of this study was to exploit loop-mediated isothermal amplification (LAMP) for the detection of SARS-CoV-2 and evaluate its performance by comparison with rRT-PCR.\n\nMethods: Reverse-transcription LAMP (RT-LAMP) assay primers were designed to detect envelop (E) and nucleocapsid (N) genes of SARS-CoV-2. Positive control RNA was prepared by in vitro transcription of E and N genes clones. RT-LAMP amplification reactions were incubated at 65\u00b0C for 30 min. Results were recorded visually. RT-LAMP results were evaluated by comparing the results obtained with a commercial rRT-PCR kit.\n\nResults: The RT-LAMP assay for E and N genes was carried out in separate tubes. RT-LAMP detected about 40 copies of SARS-CoV-2 RNA per reaction. A total of 253 throat swabs were tested using the RT-LAMP assay. The overall diagnostic sensitivity and specificity of the LAMP assay were 98.46 and 100 percent, respectively, as compared to the rRT-PCR.\n\nInterpretation & conclusions: SARS-CoV-2 RT-LAMP assay was designed, standardized and evaluated. The assay showed diagnostic sensitivity and specificity equivalent to rRT-PCR assays. The assay will be useful to increase testing capacity for the detection of SARS-CoV-2 in the country.","lamp_id":[{"id":"LPB00546","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00547","pathogen":"SARS-CoV-2","target":"E"}]},{"id":289,"pmid":35361434,"title":"PD-LAMP smartphone detection of SARS-CoV-2 on chip","year":2022,"journal":"Analytica Chimica Acta","authors":"Ashlee J Colbert, Dong Hoon Lee, Katherine N Clayton, Steven T Wereley, Jacqueline C Linnes, Tamara L Kinzer-Ursem","doi":"10.1016\/j.aca.2022.339702","country":"USA","institute":"Purdue University","deparment":"Weldon School of Biomedical Engineering","abstract":"In 2019 the COVID-19 pandemic, caused by SARS-CoV-2, demonstrated the urgent need for rapid, reliable, and portable diagnostics. The COVID-19 pandemic was declared in January 2020 and surges of the outbreak continue to reoccur. It is clear that early identification of infected individuals, especially asymptomatic carriers, plays a huge role in preventing the spread of the disease. The current gold standard diagnostic for SARS-CoV-2 is quantitative reverse transcription polymerase chain reaction (qRT-PCR) test based on the detection of the viral RNA. While RT-PCR is reliable and sensitive, it requires expensive centralized equipment and is time consuming (\u223c2 h or more); limiting its applicability in low resource areas. The FDA issued Emergency Use Authorizations (EUAs) for several COVID-19 diagnostics with an emphasis on point-of care (PoC) testing. Numerous RT-PCR and serological tests were approved for use at the point of care. Abbott's ID NOW, and Cue Health's COVID-19 test are of particular interest, which use isothermal amplification methods for rapid detection in under 20 min. We look to expand on the range of current PoC testing platforms with a new rapid and portable isothermal nucleic acid detection device. We pair reverse transcription loop mediated isothermal amplification (RT-LAMP) with a particle imaging technique, particle diffusometry (PD), to successfully detect SARS-CoV-2 in only 35 min on a portable chip with integrated heating. A smartphone device is used to image the samples containing fluorescent beads post-RT-LAMP and correlates decreased diffusivity to positive samples. We detect as little as 30 virus particles per \u03bcL from a RT-LAMP reaction in a microfluidic chip using a portable heating unit. Further, we can perform RT-LAMP from a diluted unprocessed saliva sample without RNA extraction. Additionally, we lyophilize SARS-CoV-2-specific RT-LAMP reactions that target both the N gene and the ORF1ab gene in the microfluidic chip, eliminating the need for cold storage. Our assay meets specific target product profiles outlined by the World Health Organization: it is specific to SARS-CoV-2, does not require cold storage, is compatible with digital connectivity, and has a detection limit of less than 35 \u00d7 10^4 viral particles per mL in saliva. PD-LAMP is rapid, simple, and attractive for screening and use at the point of care.","lamp_id":[{"id":"LPB00548","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"},{"id":"LPB00549","pathogen":"SARS-CoV-2","target":"N"}]},{"id":290,"pmid":35312732,"title":"Detection of SARS-CoV-2 and the L452R spike mutation using reverse transcription loop-mediated isothermal amplification plus bioluminescent assay in real-time (RT-LAMP-BART)","year":2022,"journal":"PLoS One","authors":"Takahiro Iijima, Shinnosuke Ando, Dai Kanamori, Kazumichi Kuroda, Tsutomu Nomura, Laurence Tisi, Paul E Kilgore, Neil Percy, Hikaru Kohase, Satoshi Hayakawa, Mitsuko Seki, Tomonori Hoshino","doi":"10.1371\/journal.pone.0265748","country":"Japan","institute":"Meikai University School of Dentistry","deparment":"Department of Human Development and Fostering","abstract":"The new coronavirus infection (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be fatal, and several variants of SARS-CoV-2 with mutations of the receptor-binding domain (RBD) have increased avidity for human cell receptors. A single missense mutation of U to G at nucleotide position 1355 (U1355G) in the spike (S) gene changes leucine to arginine (L452R) in the spike protein. This mutation has been observed in the India and California strains (B.1.617 and B.1.427\/B.1.429, respectively). Control of COVID-19 requires rapid and reliable detection of SARS-CoV-2. Therefore, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay plus a bioluminescent assay in real-time (BART) to detect SARS-CoV-2 and the L452R spike mutation. The specificity and sensitivity of the RT-LAMP-BART assay was evaluated using synthetic RNAs including target sequences and RNA-spiked clinical nasopharyngeal and saliva specimens as well as reference strains representing five viral and four bacterial pathogens. The novel RT-LAMP-BART assay to detect SARS-CoV-2 was highly specific compared to the conventional real-time RT-PCR. Within 25 min, the RT-LAMP-BART assay detected 80 copies of the target gene in a sample, whereas the conventional real-time RT-PCR method detected 5 copies per reaction within 130 min. Using RNA-spiked specimens, the sensitivity of the RT-LAMP-BART assay was slightly attenuated compared to purified RNA as a template. The results were identical to those of the conventional real-time RT-PCR method. Furthermore, using a peptide nucleic acid (PNA) probe, the RT-LAMP-BART method correctly identified the L452R spike mutation. This is the first report describes RT-LAMP-BART as a simple, inexpensive, rapid, and useful assay for detection of SARS-CoV-2, its variants of concern, and for screening of COVID-19.","lamp_id":[{"id":"LPB00550","pathogen":"SARS-CoV-2","target":"RdRp"},{"id":"LPB00551","pathogen":"SARS-CoV-2 (Delta)","target":"S"}]},{"id":291,"pmid":35830452,"title":"A quantitative RT-qLAMP for the detection of SARS-CoV-2 and human gene in clinical application","year":2022,"journal":"Microbial Biotechnology","authors":"Yejiong Yu, Johnny X Y Zhou, Binbin Li, Mengmeng Ji, Yun Wang, Emma Carnaby, Monique I Andersson, Wei E Huang, Zhanfeng Cui","doi":"10.1111\/1751-7915.14112","country":"United Kingdom","institute":"Institute of Biomedical Engineering","deparment":"Department of Engineering Science","abstract":"Reverse transcription (RT) - loop-mediated isothermal amplification (LAMP) assay is a rapid and one-step method to detect SARS-CoV-2 in the pandemic. Quantitative estimation of the viral load of SARS-CoV-2 in patient samples could help physicians make decisions on clinical treatment and patient management. Here, we propose to use a quantitative LAMP (qLAMP) method to evaluate the viral load of SARS-CoV-2 in samples. We used threshold time (TT) values of qLAMP, the isothermal incubation time required for the fluorescent or colorimetric signal to reach the threshold, to indicate the viral load of clinical samples. Similar to the cycle threshold (Ct ) values in conventional qPCR, TT values of qLAMP show a linear relationship to the copy numbers of SARS-CoV-2. The higher the viral loadings, the lower qLAMP TT values are. The RT-qLAMP assay was demonstrated to quantify the viral loads of synthesized full-length RNA, inactivated viral particles (BBIBP-CorV), and clinical samples within 15 min by fluorescent reading and 25 min by colorimetric reading. The RT-qLAMP has been applied to detect Alpha, Beta, Kappa, Delta, and Omicron variants of SARS-CoV-2, as well as the human beta-actin gene, and their TT values showed the linear patterns. The RT-qLAMP assays were evaluated by 64 clinical samples (25 positives and 39 negatives) for the assessment of viral loads, and it was also used to quantify the human beta-actin gene, which was used as a control and an indicator of sampling quality in clinical swab samples. The result of RT-qLAMP was in good agreement with the result of RT-qPCR. The RT-qLAMP assay detected all clinical samples, including those with Ct = 35, within 10 min using fluorescent reading.","lamp_id":[{"id":"LPB00552","pathogen":"SARS-CoV-2","target":"ORF1ab"}]},{"id":292,"pmid":35624625,"title":"All-in-One Digital Microfluidics System for Molecular Diagnosis with Loop-Mediated Isothermal Amplification","year":2022,"journal":"Biosensors","authors":"Siyi Hu, Yuhan Jie, Kai Jin, Yifan Zhang, Tianjie Guo, Qi Huang, Qian Mei, Fuqiang Ma, Hanbin Ma","doi":"10.3390\/bios12050324","country":"China","institute":"Suzhou Institute of Biomedical Engineering and Technology","deparment":"CAS Key Laboratory of Bio-Medical Diagnostics","abstract":"In this study, an \"all-in-one\" digital microfluidics (DMF) system was developed for automatic and rapid molecular diagnosis and integrated with magnetic bead-based nucleic acid extraction, loop-mediated isothermal amplification (LAMP), and real-time optical signal monitoring. First, we performed on- and off-chip comparison experiments for the magnetic bead nucleic acid extraction module and LAMP amplification function. The extraction efficiency for the on-chip test was comparable to that of conventional off-chip methods. The processing time for the automatic on-chip workflow was only 23 min, which was less than that of the conventional methods of 28 min 45 s. Meanwhile, the number of samples used in on-chip experiments was significantly smaller than that used in off-chip experiments; only 5 \u00b5L of E. coli samples was required for nucleic acid extraction, and 1 \u00b5L of the nucleic acid template was needed for the amplification reaction. In addition, we selected SARS-CoV-2 nucleic acid reference materials for the nucleic acid detection experiment, demonstrating a limit of detection of 10 copies\/\u00b5L. The proposed \"all-in-one\" DMF system provides an on-site \"sample to answer\" time of approximately 60 min, which can be a powerful tool for point-of-care molecular diagnostics.","lamp_id":[{"id":"LPB00554","pathogen":"SARS-CoV-2","target":"N"}]},{"id":293,"pmid":36040863,"title":"Paper Device Combining CRISPR\/Cas12a and Reverse-Transcription Loop-Mediated Isothermal Amplification for SARS-CoV-2 Detection in Wastewater","year":2022,"journal":"Environmental Science & Technology","authors":"Haorui Cao, Kang Mao, Fang Ran, Pengqi Xu, Yirong Zhao, Xiangyan Zhang, Hourong Zhou, Zhugen Yang, Hua Zhang, Guibin Jiang","doi":"10.1021\/acs.est.2c04727","country":"China","institute":"Institute of Geochemistry","deparment":"State Key Laboratory of Environmental Geochemistry","abstract":"Wastewater-based surveillance of the COVID-19 pandemic holds great promise; however, a point-of-use detection method for SARS-CoV-2 in wastewater is lacking. Here, a portable paper device based on CRISPR\/Cas12a and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with excellent sensitivity and specificity was developed for SARS-CoV-2 detection in wastewater. Three primer sets of RT-LAMP and guide RNAs (gRNAs) that could lead Cas12a to recognize target genes via base pairing were used to perform the high-fidelity RT-LAMP to detect the N, E, and S genes of SARS-CoV-2. Due to the trans-cleavage activity of CRISPR\/Cas12a after high-fidelity amplicon recognition, carboxyfluorescein-ssDNA-Black Hole Quencher-1 and carboxyfluorescein-ssDNA-biotin probes were adopted to realize different visualization pathways via a fluorescence or lateral flow analysis, respectively. The reactions were integrated into a paper device for simultaneously detecting the N, E, and S genes with limits of detection (LODs) of 25, 310, and 10 copies\/mL, respectively. The device achieved a semiquantitative analysis from 0 to 310 copies\/mL due to the different LODs of the three genes. Blind experiments demonstrated that the device was suitable for wastewater analysis with 97.7% sensitivity and 82% semiquantitative accuracy. This is the first semiquantitative endpoint detection of SARS-CoV-2 in wastewater via different LODs, demonstrating a promising point-of-use method for wastewater-based surveillance.","lamp_id":[{"id":"LPB00555","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00556","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00557","pathogen":"SARS-CoV-2","target":"S"}]},{"id":294,"pmid":35766650,"title":"Molecular detection of omicron SARS-CoV-2 variant is achieved by RT-LAMP despite genomic mutations","year":2022,"journal":"Mem\u00f3rias do Instituto Oswaldo Cruz","authors":"Let\u00edcia Trindade Almeida, Amanda Bonoto Gon\u00e7alves, Ana Paula Moreira Franco-Luiz, Thais B\u00e1rbara de Souza Silva, Pedro Augusto Alves, Rubens Lima do Monte-Neto","doi":"10.1590\/0074-02760220050","country":"Brazil","institute":"Instituto Ren\u00e9 Rachou","deparment":"Funda\u00e7\u00e3o Oswaldo Cruz-Fiocruz","abstract":"Background: Severe acute respiratory syndrome coronavirus (SARS-CoV-2) omicron variant was first detected in South Africa in November 2021. Since then, the number of cases due to this variant increases enormously every day in different parts of the world. Mutations within omicron genome may impair the molecular detection resulting in false negative results during Coronavirus disease 19 (COVID-19) diagnosis.\n\nObjectives: To verify if colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting N and E genes would work efficiently to detect omicron SARS-CoV-2 variant and its sub-lineages.\n\nMethods: SARS-CoV-2 reverse transcription quantitative polymerase chain reaction (RT-qPCR) positive samples were sequenced by next generation DNA sequencing. The consensus sequences generated were submitted to Pangolin tool for SARS-CoV-2 lineage identification. RT-LAMP reactions were performed at 65\u00baC\/30 min targeting N and E.\n\nFindings: SARS-CoV-2 omicron can be detected by RT-LAMP targeting N and E genes despite the genomic mutation of this more transmissible lineage. Omicron SARS-CoV-2 sub-lineages were tested and efficiently detected by RT-LAMP. We demonstrated that this test is very sensitive in detecting omicron variant, with LoD as low as 0.4 copies\/\u00b5L.\n\nMain conclusions: Molecular detection of omicron SARS-CoV-2 variant and its sub-lineages can be achieved by RT-LAMP despite the genomic mutations as a very sensitive surveillance tool for COVID-19 molecular diagnosis.","lamp_id":[{"id":"LPB00558","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00559","pathogen":"SARS-CoV-2","target":"E"}]},{"id":295,"pmid":35402391,"title":"High Sensitivity, Rapid Detection of Virus in High Traffic Environments","year":2022,"journal":"Frontiers in Bioengineering and Biotechnology","authors":"Lauren Waller, Zhilin Guo, Rui Tang, Zunming Zhang, Edward Wang, Jarred Yasuhara-Bell, Louise Laurent, Yu-Hwa Lo","doi":"10.3389\/fbioe.2022.877603","country":"USA","institute":"University of California","deparment":"Department of Bioengineering","abstract":"The global pandemic caused by the SARS-CoV-2 virus has underscored the need for rapid, simple, scalable, and high-throughput multiplex diagnostics in non-laboratory settings. Here we demonstrate a multiplex reverse-transcription loop-mediated isothermal amplification (RT-LAMP) coupled with a gold nanoparticle-based lateral flow immunoassay (LFIA) capable of detecting up to three unique viral gene targets in 15 min. RT-LAMP primers associated with three separate gene targets from the SARS-CoV-2 virus (Orf1ab, Envelope, and Nucleocapsid) were added to a one-pot mix. A colorimetric change from red to yellow occurs in the presence of a positive sample. Positive samples are run through a LFIA to achieve specificity on a multiplex three-test line paper assay. Positive results are indicated by a characteristic crimson line. The device is almost fully automated and is deployable in any community setting with a power source.","lamp_id":[{"id":"LPB00560","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00561","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00562","pathogen":"SARS-CoV-2","target":"E"}]},{"id":296,"pmid":35882427,"title":"An improved, simple and field deployable CRISPR-Cas12a assay for detection of SARS-CoV-2","year":2022,"journal":"Journal of Applied Microbiology","authors":"Chitra S Misra, Shyam Sunder Rangu, Ravindra D Phulsundar, Gargi Bindal, Mandeep Singh, R Shashidhar, T K Saha, A V S S N Rao, Devashish Rath","doi":"10.1111\/jam.15737","country":"India","institute":"Bhabha Atomic Research Centre","deparment":"Applied Genomics Section","abstract":"Aims: The RT-PCR is the most popular confirmatory test for SARS-CoV-2. It is sensitive, but high instrumentation cost makes it difficult for use outside routine clinical setup. This has necessitated the development of alternative methods such as CRISPR-based DETECTR method which uses lateral flow technology. Although accurate and sensitive, this method is limited by complex steps and recurrent cost of high-quality lateral flow strips. The main goal of this study was to improve the Cas12a-based SARS-CoV-2 DETECTR method and develop a portable and field-deployable system to reduce the recurring consumable cost.\n\nMethods and results: Specific regions of N and E genes from SARS-CoV-2 virus and human RNase P (internal control) were reverse transcribed (RT) and amplified by loop-mediated isothermal amplification (LAMP). The amplified products were detected by a Cas12a-based trans-cleavage reaction that generated a fluorescent signal which could be easily visualized by naked eye. Detection of internal control, RNase P gene was improved and optimized by redesigning RT-LAMP primers. A number of steps were reduced by combining the reagents related to the detection of Cas12a trans-cleavage reaction into a single ready-to-use mix. A portable, cost-effective battery-operated instrument, CRISPR-CUBE was developed to run the assay and visualize the outcome. The method and instrument were validated using both contrived and patient samples.\n\nConclusions: The simplified CRISPR-based SARS-CoV-2 detection and instrument developed in this study, along with improved design for internal control detection allows for easier, more definitive viral detection requiring only reagents, consumables and the battery operable CRISPR-CUBE.","lamp_id":[{"id":"LPB00563","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00564","pathogen":"SARS-CoV-2","target":"N"}]},{"id":297,"pmid":35545967,"title":"Isothermal amplification using sequence-specific fluorescence detection of SARS coronavirus 2 and variants in nasal swabs","year":2022,"journal":"Biotechniques","authors":"Les Jones, Hemant K Naikare, Yung-Yi C Mosley, Ralph A Tripp","doi":"10.2144\/btn-2022-0037","country":"USA","institute":"University of Georgia","deparment":"Department of Infectious Disease","abstract":"Coronavirus disease 2019 is a public health challenge requiring rapid testing for the detection of infections and transmission. Nucleic acid amplification tests targeting SARS coronavirus 2 (CoV2) are used to detect CoV2 in clinical samples. Real-time reverse transcription quantitative PCR is the standard nucleic acid amplification test for CoV2, although reverse transcription loop-mediated isothermal amplification is used in diagnostics. The authors demonstrate a sequence-specific reverse transcription loop-mediated isothermal amplification-based nucleic acid amplification assay that is finished within 30 min using minimally processed clinical nasal swab samples and describe a fluorescence-quenched reverse transcription loop-mediated isothermal amplification assay using labeled primers and a quencher oligonucleotide. This assay can achieve rapid (30 min) and sensitive (1000 plaque-forming units\/ml) fluorescence detection of CoV2 (WA1\/2020), B.1.1.7 (Alpha) and variants of concern Delta (B.1.617.2) and Omicron (B.1.1.529) in nasal samples.","lamp_id":[{"id":"LPB00565","pathogen":"SARS-CoV-2","target":"N"}]},{"id":298,"pmid":35909465,"title":"Inhibition of Non-specific Amplification in Loop-Mediated Isothermal Amplification via Tetramethylammonium Chloride","year":2022,"journal":"Biochip Journal","authors":"MinJu Jang, Sanghyo Kim","doi":"10.1007\/s13206-022-00070-3","country":"Republic of Korea","institute":"Gachon University","deparment":"Department of Bionanotechnology","abstract":"Loop-mediated isothermal amplification (LAMP) may be used in molecular and point-of-care diagnostics for pathogen detection. The amplification occurs under isothermal conditions using up to six primers. However, non-specific amplification is frequently observed in LAMP. Non-specific amplification has the potential to be triggered by forward and reverse internal primers. And the relatively low reaction temperature (55-65 \u00b0C) induces the secondary structure via primer-primer interactions. Primer redesign and probe design have been recommended to solve this problem. LAMP primers have strict conditions, such as Tm, GC contents, primer dimer, and distance between primers compared to conventional PCR primers. Probe design requires specialized knowledge to have high specificity for a target. In polymerase chain reaction (PCR), some chemicals or proteins are used for improving specificity and efficiency. Therefore, we hypothesized that additives can suppress the non-specific amplification. In this study, tetramethylammonium chloride (TMAC), formamide, dimethyl sulfoxide, Tween 20, and bovine serum albumin have been used as LAMP additives. In our study, TMAC was presented as a promising additive for suppressing non-specific amplification in LAMP.","lamp_id":[{"id":"LPB00566","pathogen":"SARS-CoV-2","target":"RdRp"}]},{"id":299,"pmid":19515543,"title":"A novel and more sensitive loop-mediated isothermal amplification assay targeting IS6110 for detection of Mycobacterium tuberculosis comple","year":2010,"journal":"Microbiological Research","authors":"Ehsan Aryan, Manoochehr Makvandi, Ahmad Farajzadeh, Kris Huygen, Pablo Bifani, Seyed-Latif Mousavi, Abolfazl Fateh, Abbass Jelodar, Mohammad-Mehdi Gouya, Marta Romano","doi":"10.1016\/j.micres.2009.05.001","country":"Iran","institute":"Ahvaz Jondishapour University of Medical Sciences","deparment":"Department of Medical Microbiology","abstract":"Developing improved tuberculosis (TB) diagnostics is one of the international research priorities, as TB remains globally a major health threat. Loop-mediated isothermal amplification (LAMP) is a new nucleic acid detection method that can be used in low-resource settings, because it does not require expensive or complex instruments. Using the repetitive insertion sequence IS6110 as a target gene, we developed an efficient LAMP assay, which specifically detects members of the Mycobacterium tuberculosis complex (MTBC). This assay proved 20 times more sensitive than IS6110-based conventional PCR. Moreover, its sensitivity was, respectively, 50 and 20 times higher than the one obtained with the two previously described LAMP assays for M. tuberculosis, based on gyrB and rrs, respectively. Identical sensitivities were obtained for LAMP and nested PCR, but the LAMP assay was more rapid and cost-effective than the latter. Although, our LAMP assay can successfully be performed using a non-denatured template, this results in a 200-fold reduction in the sensitivity of the assay. Moreover, by performing our LAMP assay on 15 clinical sputum samples from TB patients we were able to detect MTB. Taken together, our preliminary results indicate that IS6110-based MTBC-LAMP assay is a promising new TB-diagnostic test, with high sensitivity and that could easily be applied for the diagnosis of TB in a low-resource setting.","lamp_id":[{"id":"LPB00567","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"}]},{"id":300,"pmid":18349362,"title":"Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients","year":2008,"journal":"Journal of Medical Microbiology","authors":"Basu Dev Pandey, Ajay Poudel, Tomoko Yoda, Aki Tamaru, Naozumi Oda, Yukari Fukushima, Binod Lekhak, Basista Risal, Bishnu Acharya, Bishwa Sapkota, Chie Nakajima, Tooru Taniguchi, Benjawan Phetsuksiri, Yasuhiko Suzuki","doi":"10.1099\/jmm.0.47499-0","country":"Nepal","institute":"Everest International Clinic and Research Center\/Sukraraj Tropical and Infectious Disease Hospital","deparment":"","abstract":"A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96\/96), and the specificity in culture-negative samples was 94.2 % (98\/104, 95 % confidence interval 90.5-97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 %, respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB.","lamp_id":[{"id":"LPB00568","pathogen":"Mycobacterium tuberculosis","target":"16S rRNA"}]},{"id":301,"pmid":20071841,"title":"Clinical profiling and use of loop-mediated isothermal amplification assay for rapid detection of Mycobacterium tuberculosis from sputum","year":2009,"journal":"Kathmandu University Medical Journal","authors":"A Poudel, B D Pandey, B Lekhak, B Rijal, B R Sapkota, Y Suzuki","doi":"10.3126\/kumj.v7i2.2701","country":"Nepal","institute":"Kathmandu Medical College","deparment":"Department of Microbiology","abstract":"Background: Tuberculosis is a global health problem and the situation is worsening with newer incidences of drug resistance and HIV association. Diagnosis of tuberculosis can be done by many methods and test, culture of sputum being the ideal one. Nucleic acid amplification (NAA) assay are more time efficient one, that amplify and detect specific nucleic acid sequences allows rapid, sensitive and specific detection of M. tuberculosis in sputum samples.\n\nObjectives: The present study intends to compile the clinical presentations of the pulmonary tuberculosis (PTB) patients and to evaluate the efficacy of in-house loop-mediated isothermal amplification (LAMP) in detecting Mycobacterium tuberculosis in sputum samples by comparing with microscopy and culture.\n\nMaterials and methods: Two hundred two sputum samples were collected from 202 patients at National Tuberculosis Center, Bhaktapur, Nepal. Complete clinical profiling, epidemiological data and record on BCG vaccination were noted and the samples were subjected for microscopy, culture and in-house LAMP with six primers specific for 16S RNA gene of Mycobacterium tuberculosis.\n\nResult: Of the 176 cases of clinical profiling, productive cough was most common symptom in 147 (83.52%), followed by chest pain 136 (77.27%), fever 133 (75.56%) and haemoptysis 61 (34.66%). There was a statistically significant association between BCG vaccination and development of TB (chi(2)=5.33, P=0.02). Of 202 cases, 115 (56.93%) were chest X-ray positive, 101(50%) were direct smear-positive and 100 (49.51%) were culture positive. LAMP had a sensitivity of 97% and specificity of 94.12% while comparing with culture. In addition, its sensitivity and specificity were 91.09% and 89.11% respectively with reference to microscopy.\n\nConclusion: As in our previous study, overall, the result of present study further confirms that the in-house LAMP is a simple, rapid, sensitive and specific DNA amplification technique for PTB diagnosis. Because of rapidity of microscopy and specificity of culture, in-house LAMP assay can be used as a very powerful and useful supplementary tool with complete clinical profiling of the patients for rapid diagnosis of TB in both AFB-positive and negative cases who are suspected as PTB in disease endemic country like Nepal.","lamp_id":[{"id":"LPB00569","pathogen":"Mycobacterium tuberculosis","target":"16S rRNA"}]},{"id":302,"pmid":19616589,"title":"Use of visual loop-mediated isotheral amplification of rimM sequence for rapid detection of Mycobacterium tuberculosis and Mycobacterium bovis","year":2009,"journal":"Journal of Microbiological Methods","authors":"Ru-Yi Zhu, Kou-Xing Zhang, Ming-Qiu Zhao, Ya-Hong Liu, Yun-Yi Xu, Chun-Mei Ju, Bing Li, Jin-Ding Chen","doi":"10.1016\/j.mimet.2009.07.006","country":"China","institute":"South China Agricultural University","deparment":"College of Veterinary Medicine","abstract":"Mycobacterium tuberculosis and Mycobacterium bovis are pathogenic bacterial species in the genus Mycobacterium and the causative agents of most cases of tuberculosis (TB). Detection of M. tuberculosis and M. bovis using conventional culture- and biochemical-based assays is time-consuming and laborious. Therefore, a simple and sensitive method for rapid detection has been anxiously awaited. In the present study, a visual loop-mediated isothermal amplification (LAMP) assay was designed from the rimM (encoding 16S rRNA-processing protein) gene sequence and used to rapidly detect M. tuberculosis and M. bovis from clinical samples in South China. The visual LAMP reaction was performed by adding calcein and manganous ion, allowing the results to be read by simple visual observation of color change in a closed-tube system, and which takes less than 1 h at 65 degrees C. The assay correctly identified 84 M. tuberculosis isolates, 3 M. bovis strains and 1 M. bovis BCG samples, but did not detect 51 non-tuberculous mycobacteria (NTM) isolates and 8 other bacterial species. Sensitivity of this assay for detection of genomic DNA was 1 pg. Specific amplification was confirmed by the ladder-like pattern of gel electrophoresis and restriction enzyme HhaI digestion. The assay successfully detected M. tuberculosis and M. bovis not only in pure bacterial culture but also in clinical samples of sputum, pleural fluid and blood. The speed, specificity, sensitivity of the rimM LAMP, the lack of a need for expensive equipment, and the visual readout show great potential for clinical detection of M. tuberculosis and M. bovis.","lamp_id":[{"id":"LPB00570","pathogen":"Mycobacterium tuberculosis H37Rv","target":"rimM"}]},{"id":303,"pmid":21695047,"title":"Comparison of the efficacies of loop-mediated isothermal amplification, fluorescence smear microscopy and culture for the diagnosis of tuberculosis","year":2011,"journal":"PLoS One","authors":"Geojith George, Prem Mony, John Kenneth","doi":"10.1371\/journal.pone.0021007","country":"India","institute":"St John's Research Institute","deparment":"Division of Infectious Disease","abstract":"Background: Despite the advent of novel diagnostic techniques, smear microscopy remains as the most practical test available in resource-limited settings for tuberculosis (TB) diagnosis. Due to the low sensitivity of microscopy and the long time required for culture, feasible and accessible rapid diagnostic methods are urgently needed. Loop-mediated Isothermal Amplification (LAMP) is a promising nucleic-acid amplification assay, which could be accessible, cost-effective and more suited for use with unpurified samples.\n\nMethodology\/principal findings: In the current study, the objective was to assess the efficacy of a LAMP assay for tuberculosis compared with fluorescence smear microscopy as well as L\u00f6wenstein-Jensen (LJ) and Mycobacteria Growth Indicator Tube (MGIT) cultures for the diagnosis of pulmonary tuberculosis using sputum samples. Smear microscopy and culture were performed for decontaminated and concentrated sputum from TB suspects and the LAMP was also performed on these specimens. The LAMP and smear microscopy were compared, in series and in parallel, to culture. LAMP and smear microscopy showed sensitivities of 79.5% and 82.1% respectively and specificities of 93.8% and 96.9% respectively, compared to culture. LAMP and smear in series had sensitivity and specificity of 79.5% and 100.0% respectively. LAMP and smear in parallel had sensitivity and specificity of 82.1% and 90.6% respectively.\n\nConclusions\/significance: The overall efficacies of LAMP and fluorescence smear microscopy in the current study were high and broadly similar. LAMP and smear in series had high specificity (100.0%) and can be used as a rule-in test combination. However, the performance of LAMP in smear negative samples was found to be insufficient.","lamp_id":[{"id":"LPB00571","pathogen":"Mycobacterium tuberculosis H37Rv","target":"rimM"}]},{"id":304,"pmid":32115540,"title":"Comparison of Loop-Mediated Isothermal Amplification, Microscopy, Culture, and PCR for Diagnosis of Pulmonary Tuberculosis","year":2020,"journal":"Japanese Journal of Infectious Diseases","authors":"Benjawan Phetsuksiri, Janisara Rudeeaneksin, Sopa Srisungngam, Supranee Bunchoo, Wiphat Klayut Chie Nakajima, Shigeyuki Hamada, Yasuhiko Suzuki","doi":"10.7883\/yoken.JJID.2019.335","country":"Thailand","institute":"National Institute of Health","deparment":"Department of Medical Sciences","abstract":"The diagnosis of tuberculosis (TB) in endemic countries is challenging due to high caseloads and limited resources. A simple and cost-effective diagnostic test for the rapid detection of Mycobacterium tuberculosis (M. tuberculosis) in clinical specimens is crucially needed. We evaluated the performance of an in-house assay based on loop-mediated isothermal amplification (LAMP) targeting the M. tuberculosis 16S ribosomal RNA (rRNA) gene for the diagnosis of TB in Thailand. A total of 252 sputum samples from suspected cases of pulmonary TB were analyzed. The sensitivity of LAMP was 99.04% (103\/104; 95% confidence interval [CI]: 94.76-9.98%) and 72.73% (16\/22; 95% CI: 49.78-89.27%) for smear-positive and smear-negative samples with TB-culture positivity, respectively. LAMP detected 20.69% (24\/116) of TB culture negative samples but all those were positive by conventional polymerase chain reaction (PCR). The sensitivity of LAMP was higher than that of sputum microscopy while the performance of LAMP was similar to PCR. None of the samples positive for non-tuberculous mycobacteria by culture and PCR were positive by LAMP. Compared to TB culture, the positive predictive value (PPV), negative predictive value (NPV), and kappa coefficient of LAMP were 83.22%, 88.33%, and 0.75 respectively. Based on the diagnostic performance, we propose that LAMP would be suitable as a potential diagnostic test for rapid TB diagnosis in resource-limited laboratory settings.","lamp_id":[{"id":"LPB00573","pathogen":"Mycobacterium tuberculosis","target":"16S rRNA"}]},{"id":305,"pmid":26218925,"title":"Clinical Specimen-Direct LAMP: A Useful Tool for the Surveillance of blaOXA-23-Positive Carbapenem-Resistant Acinetobacter baumannii","year":2015,"journal":"PLoS One","authors":"Norihisa Yamamoto, Shigeto Hamaguchi, Yukihiro Akeda, Pitak Santanirand, Anusak Kerdsin, Masafumi Seki, Yoshikazu Ishii, Wantana Paveenkittiporn, Robert A Bonomo, Kazunori Oishi, Kumthorn Malathum, Kazunori Tomono","doi":"10.1371\/journal.pone.0133204","country":"","institute":"","deparment":"","abstract":"Healthcare-associated infections are a leading cause of morbidity and mortality worldwide. Treatment is increasingly complicated by the escalating incidence of antimicrobial resistance. Among drug-resistant pathogens, carbapenem-resistant Acinetobacter baumannii (CRAb) is of increasing concern because of the limited applicable therapies and its expanding global distribution in developed countries and newly industrialized countries. Therefore, a rapid detection method that can be used even in resource-poor countries is urgently required to control this global public health threat. Conventional techniques, such as bacterial culture and polymerase chain reaction (PCR), are insufficient to combat this threat because they are time-consuming and laborious. In this study, we developed a loop-mediated isothermal amplification (LAMP) method for detecting blaOXA-23-positive CRAb, the most prevalent form of CRAb in Asia, especially in Thailand, and confirmed its efficacy as a surveillance tool in a clinical setting. Clinical samples of sputum and rectal swabs were collected from patients in a hospital in Bangkok and used for LAMP assays. After boiling and centrifugation, the supernatants were used directly in the assay. In parallel, a culture method was used for comparison purposes to evaluate the specificity and sensitivity of LAMP. As a first step, a total of 120 sputum samples were collected. The sensitivity of LAMP was 88.6% (39\/44), and its specificity was 92.1% (70\/76) using the culture method as the \"gold standard\". When surveillance samples including sputum and rectal swabs were analyzed with the LAMP assay, its sensitivity was 100.0%. This method enables the direct analysis of clinical specimens and provides results within 40 minutes of sample collection, making it a useful tool for surveillance even in resource-poor countries.","lamp_id":[{"id":"LPB00574","pathogen":"Acinetobacter baumannii","target":"bla OXA-23"}]},{"id":306,"pmid":33788361,"title":"Simple and rapid detection Aspergillus fumigatus by loop-mediated isothermal amplification coupled with lateral flow biosensor assay","year":2021,"journal":"Journal of Applied Microbiology","authors":"L Jiang, R Gu, X Li, D Mu","doi":"10.1111\/jam.15092","country":"","institute":"","deparment":"","abstract":"Aims: We have developed a new diagnostic technique, termed loop-mediated isothermal amplification coupled with lateral flow biosensor (LAMP-LFB), which has been successfully applied to the detection of Aspergillus fumigatus.\n\nMaterial and methods: A set of six LAMP primers was designed according to the A. fumigatus-specific anxC4 gene, which specifically recognized eight different regions of the target sequence. The LFB was employed for reporting the A. fumigatus-LAMP results, and the visual readouts were obtained within 2 min. The strains of A. fumigatus species and non-A. fumigatus species were used to test the assay's sensitivity and examine the analytical specificity of the target assay. Optimal LAMP conditions were 66\u00b0C for 50 min. The limit of detection is 100 fg. No cross-reactions were obtained, and the specificity of LAMP-LFB assay was 100%. The whole process of the assay, including 20 min of DNA preparation, 50 min of constant temperature amplification, and 2 min of detection by the sensor strip, took a total of 72 min (less than 75 min). Among 89 sputum specimens for clinical evaluation, 10 (11\u00b723%) samples were A. fumigatus-positive by LAMP-LFB and traditional culture method, 9 (10\u00b711%) samples were A. fumigatus-positive by PCR method. Compared with culture method, the diagnostic accuracy of LAMP-LFB method was 100%.\n\nConclusions: The novel LAMP-LFB detection technology established in the current research is a rapid and reliable detection tool for A. fumigatus.\n\nSignificance and impact of the study: This novel LAMP-LFB assay can quickly, specifically and sensitively detect A. fumigatus, thereby speeding up the detection process and increasing the detection rate. In addition, it can also be used as a new molecular method for detection of A. fumigatus in clinical and laboratory areas.","lamp_id":[{"id":"LPB00575","pathogen":"Aspergillus fumigatus","target":"anxC4"}]},{"id":307,"pmid":32907990,"title":"Rapid Detection of Azole-Resistant Aspergillus fumigatus in Clinical and Environmental Isolates by Use of a Lab-on-a-Chip Diagnostic System","year":2020,"journal":"Journal of Clinical Microbiology","authors":"Ling-Shan Yu, Jesus Rodriguez-Manzano, Nicolas Moser, Ahmad Moniri, Kenny Malpartida-Cardenas, Nicholas Miscourides, Thomas Sewell, Tatiana Kochina, Amelie Brackin, Johanna Rhodes, Alison H Holmes, Matthew C Fisher, Pantelis Georgiou","doi":"10.1128\/JCM.00843-20","country":"","institute":"","deparment":"","abstract":"Aspergillus fumigatus has widely evolved resistance to the most commonly used class of antifungal chemicals, the azoles. Current methods for identifying azole resistance are time-consuming and depend on specialized laboratories. There is an urgent need for rapid detection of these emerging pathogens at point-of-care to provide the appropriate treatment in the clinic and to improve management of environmental reservoirs to mitigate the spread of antifungal resistance. Our study demonstrates the rapid and portable detection of the two most relevant genetic markers linked to azole resistance, the mutations TR34 and TR46, found in the promoter region of the gene encoding the azole target cyp51A. We developed a lab-on-a-chip platform consisting of: (i) tandem-repeat loop-mediated isothermal amplification; (ii) state-of-the-art complementary metal-oxide-semiconductor microchip technology for nucleic acid amplification detection; and (iii) a smartphone application for data acquisition, visualization, and cloud connectivity. Specific and sensitive detection was validated with isolates from clinical and environmental samples from 6 countries across 5 continents, showing a lower limit of detection of 10 genomic copies per reaction in less than 30 min. When fully integrated with a sample preparation module, this diagnostic system will enable the detection of this ubiquitous fungus at the point-of-care, and could help to improve clinical decision making, infection control, and epidemiological surveillance.","lamp_id":[{"id":"LPB00576","pathogen":"Aspergillus fumigatus","target":"cyp51A"}]},{"id":308,"pmid":27984058,"title":"Enhancing melting curve analysis for the discrimination of loop-mediated isothermal amplification products from four pathogenic molds: Use of inorganic pyrophosphatase and its effect in reducing the variance in melting temperature values","year":2017,"journal":"Journal of Microbiological Methods","authors":"Kazuya Tone, Ryuichi Fujisaki, Takashi Yamazaki, Koichi Makimura","doi":"10.1016\/j.mimet.2016.10.020","country":"","institute":"","deparment":"","abstract":"Loop-mediated isothermal amplification (LAMP) is widely used for differentiating causative agents in infectious diseases. Melting curve analysis (MCA) in conjunction with the LAMP method reduces both the labor required to conduct an assay and contamination of the products. However, two factors influence the melting temperature (Tm) of LAMP products: an inconsistent concentration of Mg2+ ion due to the precipitation of Mg2P2O7, and the guanine-cytosine (GC) content of the starting dumbbell-like structure. In this study, we investigated the influence of inorganic pyrophosphatase (PPase), an enzyme that inhibits the production of Mg2P2O7, on the Tm of LAMP products, and examined the correlation between the above factors and the Tm value using MCA. A set of LAMP primers that amplify the ribosomal DNA of the large subunit of Aspergillus fumigatus, Penicillium expansum, Penicillium marneffei, and Histoplasma capsulatum was designed, and the LAMP reaction was performed using serial concentrations of these fungal genomic DNAs as templates in the presence and absence of PPase. We compared the Tm values obtained from the PPase-free group and the PPase-containing group, and the relationship between the GC content of the theoretical starting dumbbell-like structure and the Tm values of the LAMP product from each fungus was analyzed. The range of Tm values obtained for several fungi overlapped in the PPase-free group. In contrast, in the PPase-containing group, the variance in Tm values was smaller and there was no overlap in the Tm values obtained for all fungi tested: the LAMP product of each fungus had a specific Tm value, and the average Tm value increased as the GC% of the starting dumbbell-like structure increased. The use of PPase therefore reduced the variance in the Tm value and allowed the differentiation of these pathogenic fungi using the MCA method.","lamp_id":[{"id":"LPB00577","pathogen":"Aspergillus fumigatus","target":"Large subunit rRNA"}]},{"id":309,"pmid":32402994,"title":"Graphene-based electrochemical genosensor incorporated loop-mediated isothermal amplification for rapid on-site detection of Mycobacterium tuberculosis","year":2020,"journal":"Journal of Pharmaceutical and Biomedical Analysis","authors":"Wansadaj Jaroenram, Jantana Kampeera, Narong Arunrut, Chanpen Karuwan, Assawapong Sappat, Pakapreud Khumwan, Sarinya Jaitrong, Kobporn Boonnak, Therdsak Prammananan, Angkana Chaiprasert, Adisorn Tuantranont, Wansika Kiatpathomchai","doi":"10.1016\/j.jpba.2020.113333","country":"","institute":"","deparment":"","abstract":"Tuberculosis (TB) is one of the most contagious and lethal infectious diseases that affects more than 10 million individuals worldwide. A lack of rapid TB diagnosis is partly responsible for its alarming spread and prevalence in many regions. To address this problem, we report a novel integrated point-of-care platform to detect a TB-causative bacterium, Mycobacterium tuberculosis (Mtb). This leverages loop-mediated isothermal amplification (LAMP) for Mtb-DNA amplification and the screen-printed graphene electrode (SPGE) for label-free electrochemical analysis of DNA amplicons. When implemented on a portable potentiostat device developed in-house, the system (LAMP-EC) offers a rapid end-point qualitative analysis of specific DNA amplicons that will be displayed as a discrete positive\/negative readout on the LCD screen. Under optimized conditions, LAMP-EC showed a comparable detection limit to the previously developed LAMP assay with a lateral flow readout at 1 pg total DNA or 40 Mtb genome equivalents. This highly specific technique detected the presence of TB in all 104 blinded sputum samples with a 100% accuracy. Our technique can also easily be clinically adopted due to its affordability (\u223cUSD2.5\/test), rapidity (<65 min turnaround time) and feasibility (lack of advanced instrumental requirement). This serves as a practical incentive, appealing to users in both high- and low-resource settings across the TB endemic regions and economic backgrounds.","lamp_id":[{"id":"LPB00578","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"}]},{"id":310,"pmid":32525836,"title":"Development of a LAMP method for detection of carbapenem-resistant Acinetobacter baumannii during a hospital outbreak","year":2020,"journal":"Journal of Infection in Developing Countries","authors":"Carolina Garciglia Mercado, Ramon Gaxiola Robles, Felipe Ascencio, Jesus Silva-Sanchez, Maria Teresa Estrada-Garcia, Gracia Gomez-Anduro","doi":"10.3855\/jidc.11692","country":"","institute":"","deparment":"","abstract":"Introduction: Carbapenem-resistant A. baumannii (CRAB) represents a public health threat increasing worldwide. We assess the suitability of a loop-mediated isothermal amplification (LAMP) method for on-site screening of CRAB in a hospital facility.\n\nMethodology: A set of six primers were designed for recognizing eight distinct sequences on six targets: blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaIMP, and blaVIM. A LAMP method was developed, optimized and evaluated for the identification of CRAB in thirty-three environmental samples from an outbreak in an Intensive Care Unit (ICU) facility.\n\nResults: The sensitivity of the LAMP assay for the detection of A. baumannii was ten-fold higher than the PCR assay (1.0 ng.\u00b5L-1). The LAMP assays showed a higher detection rate for CRAB samples and robust diagnosis performance in comparison to a conventional PCR, with clinical sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 100% for blaOXA-23-like, blaOXA-51-like and blaVIM.\n\nConclusions: The developed LAMP assays are powerful tools that can be useful in on-site screening of CRAB causing local outbreaks in clinics and hospitals facilities where costs and equipment restraints are imperative.","lamp_id":[{"id":"LPB00579","pathogen":"Acinetobacter baumannii","target":"blaOXA-51-like"}]},{"id":311,"pmid":31378262,"title":"Direct detection of Mycobacterium tuberculosis in clinical samples by a dry methyl green loop-mediated isothermal amplification (LAMP) method","year":2019,"journal":"Tuberculosis","authors":"Jeewan Thapa, Bhagwan Maharjan, Meena Malla, Yukari Fukushima, Ajay Poudel, Basu Dev Pandey, Kyoko Hyashida, Stephen V Gordon, Chie Nakajima, Yasuhiko Suzuki","doi":"10.1016\/j.tube.2019.05.004","country":"","institute":"","deparment":"","abstract":"The purpose of this study was to develop a simple visual methyl green (MeG) based dry loop-mediated isothermal amplification (LAMP) method for early detection of Mycobacterium tuberculosis (MTB) from clinical samples. We identified MeG as an indicator of a positive LAMP reaction, where a positive reaction gave a blue-green color while a negative reaction was colorless. The MeG MTB-LAMP system was further simplified by drying all reagents for ease of use, and was then validated for its ability to diagnose TB directly using Nepalese clinical samples. We evaluated the dry MeG MTB-LAMP with 69 new TB suspected samples from patients that did not have a confirmed history of TB treatment and found the sensitivity in culture positive samples as 92.8% (13\/14) and specificity in culture negative samples as 96.3% (53\/55). Our LAMP system has the potential to be a point of care test for early diagnosis of active TB in developing countries.","lamp_id":[{"id":"LPB00580","pathogen":"Mycobacterium tuberculosis","target":"16S rRNA"}]},{"id":312,"pmid":19022304,"title":"Evaluation of reverse transcription loop-mediated isothermal amplification in conjunction with ELISA\u2013hybridization assay for molecular detection of Mycobacterium tuberculosis","year":2009,"journal":"Journal of Microbiological Methods","authors":"Mei-Feng Lee, Yen-Hsu Chen, Chien-Fang Peng","doi":"10.1016\/j.mimet.2008.10.005","country":"Taiwan","institute":"Kaohsiung Medical University","deparment":"College of Medicine","abstract":"Traditional culture, followed by a panel of biochemical tests for the diagnosis of tuberculosis (TB), is time-consuming, and rapid identification of Mycobacterium tuberculosis is crucial for the early administration of appropriate therapy. In this study, the reverse transcription loop-mediated isothermal amplification combined with enzyme-linked immunosorbent hybridization (RT-LAMP-ELISA-hybridization) assay has been designed for the rapid detection of 16S rRNA in clinical isolates of M. tuberculosis. This assay reproducibly detected a single copy, as opposed to 2000 copies of MTB 16S rRNA detected by conventional gel electrophoresis. Among the 150 specimens of sputum analysed, RT-LAMP-ELISA-hybridization assay had a sensitivity of 94.1% in the culture method, compared to the Amplified M. tuberculosis Direct Test (AMTD), 91.1% and the 88.2% sensitivity of acid-fast staining. Furthermore, RT-LAMP-ELISA-hybridization assay is more cost-effective when compared to the real-time TaqMan RT-PCR and AMTD assays. In conclusion, our results suggest that the RT-LAMP-ELISA-hybridization assay is a highly sensitive, low cost diagnostic tool useful for the rapid and accurate direct diagnosis of sputum specimens, and is suitable for routine clinical use.","lamp_id":[{"id":"LPB00581","pathogen":"Mycobacterium tuberculosis H37Rv","target":"16S rDNA"}]},{"id":313,"pmid":26707336,"title":"Loop-mediated isothermal amplification: Rapid and sensitive detection of the antibiotic resistance gene ISAba1-blaOXA-51-like in Acinetobacter baumannii","year":2016,"journal":"Journal of Microbiological Methods","authors":"Xiaoqin Mu, Ryuichi Nakano, Akiyo Nakano, Tsuneyuki Ubagai, Takane Kikuchi-Ueda, Shigeru Tansho-Nagakawa, Hirotoshi Kikuchi, Go Kamoshida, Shiro Endo, Hisakazu Yano, Yasuo Ono","doi":"10.1016\/j.mimet.2015.12.011","country":"","institute":"","deparment":"","abstract":"Carbapenem-resistant Acinetobacter baumannii, which are mainly induced by the production of OXA-type \u03b2-lactamases, are among the leading causes of nosocomial infections worldwide. Among the \u03b2-lactamase genes, the presence of the OXA-51-like gene carrying the upstream insertion sequence, ISAba1, was found to be one of the most prevalent carbapenem resistance mechanisms utilized by these bacteria. Consequently, it is necessary to develop a rapid detection method for ISAba1-blaOXA-51-like sequence for the timely and appropriate antibiotic treatment of A. baumannii infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was optimized for ISAba1-blaOXA-51-like detection. The LAMP primer set was designed to recognize distinct sequences in the ISAba1-blaOXA-51-like gene and could amplify the gene within 25 min at an isothermal temperature of 60\u00b0C. This LAMP assay was able to detect the ISAba1-blaOXA-51-like gene with high specificity; in addition, no cross-reactivity was observed for other types of \u03b2-lactamase producers (OXA-23-like, OXA-40-like, OXA-58-like, and IMP-1), as indicated by the absence of false positive or false negative results. The detection limit for this assay was found to be 10(0)CFU per tube which was 100-fold more sensitive than a polymerase chain reaction assay for ISAba1-blaOXA-51-like detection. Furthermore, the LAMP assay provided swift detection of the ISAba1-blaOXA-51-like gene, even directly from clinical specimens. In summary, we have described a new, rapid assay for the detection of the ISAba1-blaOXA-51-like gene from A. baumannii that could be useful in a clinical setting. This method might facilitate epidemiological studies and allow monitoring of the emergence of drug resistant strains.","lamp_id":[{"id":"LPB00582","pathogen":"Acinetobacter baumannii","target":"ISAba1-blaOXA-51-like"}]},{"id":314,"pmid":15814976,"title":"Rapid diagnostic method for detection of mumps virus genome by loop-mediated isothermal amplification","year":2005,"journal":"Journal of Clinical Microbiology","authors":"Takao Okafuji, Naoko Yoshida, Motoko Fujino, Yoshie Motegi, Toshiaki Ihara, Yoshinori Ota, Tsugunori Notomi, Tetsuo Nakayama","doi":"10.1128\/JCM.43.4.1625-1631.2005","country":"Japan","institute":"Okafuji Pediatric Clinic","deparment":"","abstract":"Most mumps patients are clinically diagnosed without any virological examinations, but some diagnosed cases of mumps may be caused by other pathogens or secondary vaccine failure (SVF). To clarify these issues, a sensitive, specific, and rapid diagnostic method is required. We obtained 60 salivary swabs from 34 patients with natural infection during the course of the illness, 10 samples from patients with vaccine-associated parotitis, and 5 samples from patients with SVF. Total RNA was extracted and subjected to reverse transcription-PCR (RT-PCR) and loop-mediated isothermal amplification (LAMP) for genome amplification. We detected mumps virus RNA corresponding to 0.1 PFU by LAMP within 60 min after RNA extraction, with the same sensitivity as RT-nested PCR. Mumps virus was isolated in 30 of 33 samples within day 2, and mumps virus genome was amplified by LAMP in 32 of them. The quantity of virus titer was calculated by monitoring the time to reach the threshold of turbidity. The viral load decreased after day 3 and was lower in patients serologically diagnosed as having SVF with milder illness. Accuracy of LAMP for the detection of mumps virus genome was confirmed; furthermore, it is of benefit for calculating the viral load, which reflects disease pathogenesis.","lamp_id":[{"id":"LPB00583","pathogen":"Mumps virus","target":"HN"}]},{"id":315,"pmid":18065667,"title":"Development of a loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia","year":2008,"journal":"Journal of Medical Microbiology","authors":"Natsu Uemura, Koichi Makimura, Masanobu Onozaki, Yoshihito Otsuka, Yasuhiro Shibuya, Hirohisa Yazaki, Yoshimi Kikuchi, Shigeru Abe, Shoji Kudoh","doi":"10.1099\/jmm.0.47216-0","country":"Japan","institute":"Nippon Medical School","deparment":"Department of Pulmonary Medicine\/Infection and Oncology","abstract":"Loop-mediated isothermal amplification (LAMP) is a novel, rapid nucleic acid amplification method with high specificity and sensitivity under isothermal conditions. In this study a LAMP assay for diagnosing Pneumocystis pneumonia (PCP) was developed. Oligonucleotide primers specific for Pneumocystis species were designed corresponding to 18S rRNA gene sequences. The assay, performed for 30 min at 61 degrees C, was capable of detecting 50 copies per tube (2 x 10(3) copies ml(-1)) in 30 min and did not show cross-reactivity to other species of fungi, including the genera Candida, Aspergillus and Cryptococcus. A total of 21 of 24 clinical specimens (sputum and bronchoalveolar lavage fluid) from patients with suspected PCP tested positive using the LAMP assay by real-time fluorescence detection. The results of the LAMP reaction were also observed by real-time turbidity detection and end-point visual turbidity or fluorescence detection. With real-time fluorescence detection, melting curves of the products were effective at distinguishing specific amplification from non-specific amplification or self-amplification. Visual detection was also possible as a rapid and easy assay using only a heat block and a black light.","lamp_id":[{"id":"LPB00584","pathogen":"Pneumocystis jirovecii","target":"18S rRNA"}]},{"id":316,"pmid":23220188,"title":"Rapid and sensitive detection of Acinetobacter baumannii using loop-mediated isothermal amplification","year":2013,"journal":"Journal of Microbiological Methods","authors":"Po-Chi Soo, Chun-Chieh Tseng, Siao-Ru Ling, Ming-Li Liou, Chih-Chin Liu, Huei-Jen Chao, Teng-Yi Lin, Kai-Chih Chang","doi":"10.1016\/j.mimet.2012.11.020","country":"","institute":"","deparment":"","abstract":"Here we report the design and evaluation of a loop-mediated isothermal amplification (LAMP) assay for detecting Acinetobacter baumannii DNA based on the 16S-23S rRNA intergenic spacer (ITS) sequence. The results showed that target DNA was amplified and visualized within 30min and with a detection limit 100-fold greater than PCR.","lamp_id":[{"id":"LPB00585","pathogen":"Acinetobacter baumannii","target":"ITS"}]},{"id":317,"pmid":22512310,"title":"Simple and specific detection of Bordetella holmesii by using a loop-mediated isothermal amplification assay","year":2012,"journal":"Medical Microbiology and Immunology","authors":"Nao Otsuka, Shuji Yoshino, Kimiko Kawano, Hiromi Toyoizumi-Ajisaka, Keigo Shibayama, Kazunari Kamachi","doi":"10.1111\/j.1348-0421.2012.00465.x","country":"","institute":"","deparment":"","abstract":"A loop-mediated isothermal amplification (LAMP) assay for simple detection of Bordetella holmesii was developed. This assay discriminates between B. holmesii and other Bordetella species and successfully detect B. holmesii DNA in nasopharyngeal swab samples from subjects with suspected pertussis. The LAMP assay results were in complete agreement with the results of previously published real-time PCR assay, indicating that the former is a powerful tool for the accurate diagnosis and surveillance of B. holmesii.","lamp_id":[{"id":"LPB00586","pathogen":"Bordetella holmesii","target":"recA"}]},{"id":318,"pmid":26208001,"title":"A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification","year":2015,"journal":"PLoS One","authors":"Yuji Kouzaki, Takuya Maeda, Hiroaki Sasaki, Shinsuke Tamura, Takaaki Hamamoto, Atsushi Yuki, Akinori Sato, Yasushi Miyahira, Akihiko Kawana","doi":"10.1371\/journal.pone.0133759","country":"Japan","institute":"National Defense Medical College","deparment":"Department of Internal Medicine","abstract":"Bacillus Calmette-Gu\u00e9rin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64 \u00b0C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 \u00d7 10(3) cells\/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 \u00d7 10(3) cells\/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation involving immunocompromised patients because of its convenience, rapidity, and cost effectiveness.","lamp_id":[{"id":"LPB00587","pathogen":"Mycobacterium tuberculosis BCG","target":"RD1"}]},{"id":319,"pmid":26179304,"title":"Isothermal Detection of Mycoplasma pneumoniae Directly from Respiratory Clinical Specimens","year":2015,"journal":"Journal of Clinical Microbiology","authors":"Brianna L Petrone, Bernard J Wolff, Alexandra A DeLaney, Maureen H Diaz, Jonas M Winchell","doi":"10.1128\/JCM.01431-15","country":"USA","institute":"National Center for Immunization and Respiratory Diseases","deparment":"Division of Bacterial Diseases","abstract":"Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia (CAP) across patient populations of all ages. We have developed a loop-mediated isothermal amplification (LAMP) assay that enables rapid, low-cost detection of M. pneumoniae from nucleic acid extracts and directly from various respiratory specimen types. The assay implements calcein to facilitate simple visual readout of positive results in approximately 1 h, making it ideal for use in primary care facilities and resource-poor settings. The analytical sensitivity of the assay was determined to be 100 fg by testing serial dilutions of target DNA ranging from 1 ng to 1 fg per reaction, and no cross-reactivity was observed against 17 other Mycoplasma species, 27 common respiratory agents, or human DNA. We demonstrated the utility of this assay by testing nucleic acid extracts (n = 252) and unextracted respiratory specimens (n = 72) collected during M. pneumoniae outbreaks and sporadic cases occurring in the United States from February 2010 to January 2014. The sensitivity of the LAMP assay was 88.5% tested on extracted nucleic acid and 82.1% evaluated on unextracted clinical specimens compared to a validated real-time PCR test. Further optimization and improvements to this method may lead to the availability of a rapid, cost-efficient laboratory test for M. pneumoniae detection that is more widely available to primary care facilities, ultimately facilitating prompt detection and appropriate responses to potential M. pneumoniae outbreaks and clusters within the community.","lamp_id":[{"id":"LPB00588","pathogen":"Mycoplasma pneumoniae","target":"CARDS toxin"}]},{"id":320,"pmid":26219625,"title":"Development of loop-mediated isothermal amplification assays for genotyping of Type III Secretion System in Pseudomonas aeruginosa","year":2015,"journal":"Letters in Applied Microbiology","authors":"H Shi, Z Chen, J Kan","doi":"10.1111\/lam.12469","country":"China","institute":"Southwest University","deparment":"College of Food Science","abstract":"Pseudomonas aeruginosa is a well-known environmental bacterium capable of causing a variety of life-threatening human infections, with a Type III Secretion System (T3SS) as the most significant virulence determinant. P. aeruginosa strains exhibit unique T3SS virulence genotypes defined by the presence of either exoS or exoU. In this study, loop-mediated isothermal amplification (LAMP) assays for rapid detection of exoS and exoU in P. aeruginosa have been developed and evaluated. Set of four primers were designed for LAMP-based amplification of exoS and exoU respectively. The LAMP reactions were performed at 63\u00b0C for 40 min, with detection limits of 100 fg purified DNA. In 107 river water isolates, exoS and exoU were detected in 10 (9%) and 89 (83%) isolates, respectively, and in 38 soil isolates, they were detected in 7 (18%) and 31 (82%) cases respectively. In conclusion, the LAMP assays are rapid, simple and cost-effective tools for detection of the exoU- and exoS-types of P. aeruginosa strains.","lamp_id":[{"id":"LPB00589","pathogen":"Pseudomonas aeruginosa UCBPP-PA14","target":"exoU"},{"id":"LPB00590","pathogen":"Pseudomonas aeruginosa UCBPP-PA14","target":"exoS"}]},{"id":321,"pmid":26052327,"title":"Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, China","year":2015,"journal":"Frontiers in Microbiology","authors":"Derong Dong, Wei Liu, Huan Li, Yufei Wang, Xinran Li, Dayang Zou, Zhan Yang, Simo Huang, Dongsheng Zhou, Liuyu Huang, Jing Yuan","doi":"10.3389\/fmicb.2015.00519","country":"China","institute":"Academy of Military Medical Sciences Beijing","deparment":"Institute of Disease Control and Prevention","abstract":"Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniaein clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg\/\u03bcl DNA within 60 min under isothermal conditions (61\u00b0C), a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST) groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC)-encoding gene bla KPC-2 and had high resistance to \u03b2-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain bla KPC-2 and bla NDM-1genes simultaneously in the isolate. Our data showed the high prevalence of bla KPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis and primary quarantine purposes.","lamp_id":[{"id":"LPB00591","pathogen":"Klebsiella pneumoniae subsp. pneumoniae NTUH-K2044","target":"rcsA"}]},{"id":322,"pmid":25674236,"title":"Protein-coding housekeeping gene Rv2461c can be used as an amplification target in loop-mediated isothermal amplification assay for the detection of Mycobacterium tuberculosis in sputum samples","year":2014,"journal":"International Journal of Clinical and Experimental Pathology","authors":"Dairong Li, Jianing Zhao, Xiaoping Nie, Tao Wan, Wenchun Xu, Yong Zhao","doi":"","country":"P. R. China","institute":"The First Affiliated Hospital of Chongqing Medical University Chongqing","deparment":"Department of Respiratory Medicine","abstract":"The study is to explore the potential of the conserved Rv2461c gene as a biomarker for Tuberculosis (TB) diagnosis. The conservation of the hypothetical genes was evaluated in this study using multiple sequence alignment and phylogenetic analysis. The conservation of Rv2461c coding gene was evaluated by polymerase chain reaction using six reference strains of M. tuberculosis complex (MTC), 156 M. tuberculosis clinical isolates, 25 species of non-tuberculosis mycobacteria (NTM), and 10 non-mycobacterial species. A total of 126 clinical sputum specimens were collected from patients with respiratory symptoms, including 79 specimens from suspected TB patients, and 47 specimens from patients with respiratory diseases other than TB. Genomic DNAs were extracted and subject to polymerase chain reaction for nucleic acid amplification test. In addition, we successfully developed loop-mediated isothermal amplification (LAMP) technology for rapid detection of M. tuberculosis in sputum specimens. The sensitivity and specificity of LAMP assay were evaluated for the detection of M. tuberculosis. Phylogenetic analysis of the clpP sequences revealed that the Mycobacterium strains were split into two major clusters: i) MTC; ii) NTM strains and M. leprae. During the evaluation of the conservation of Rv2461c coding gene, all MTC strains yielded positive results, and no false-positive results were observed in NTM or other bacterial species. LAMP analysis showed high sensitivity and specificity (84.8% and 95.7%, respectively) for the detection of M. tuberculosis from sputum. Our result indicated that Rv2461c coding gene was an efficient and promising alternative nucleic acid amplification test target for the detection of M. tuberculosis.","lamp_id":[{"id":"LPB00592","pathogen":"Mycobacterium tuberculosis H37Rv","target":"Rv2461c"}]},{"id":323,"pmid":25538701,"title":"Prevalence and detection of Stenotrophomonas maltophilia carrying metallo-\u03b2-lactamase blaL1 in Beijing, China","year":2014,"journal":"Frontiers in Microbiology","authors":"Zhan Yang, Wei Liu, Qian Cui, Wenkai Niu, Huan Li, Xiangna Zhao, Xiao Wei, Xuesong Wang, Simo Huang, Derong Dong, Sijing Lu, Changqing Bai, Yan Li, Liuyu Huang, Jing Yuan","doi":"10.3389\/fmicb.2014.00692","country":"China","institute":"Academy of Military Medical Sciences Beijing","deparment":"Institute of Disease Control and Prevention","abstract":"Intrinsic \u03b2-lactam resistance in Stenotrophomonas maltophilia is caused by bla L1 and\/or bla L2, a kind of metallo-\u03b2-lactamase with a broad substrate spectrum including carbapenems. A rapid and sensitive molecular method for the detection of bla L1 in clinical samples is needed to guide therapeutic treatment. In present study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of bla L1 in clinical samples by using two methods including a chromogenic method using calcein\/Mn(2+) complex and the real-time turbidity monitoring to assess the reaction. Then dissemination of L1-producing S. maltophilia was investigated from ICU patients in three top hospital in Beijing, China. The results showed that both methods detected the target DNA within 60 min under isothermal conditions (65\u00b0C). The detection limit of LAMP was 3.79 pg\/\u03bcl DNA, and its sensitivity 100-fold greater than that of conventional PCR. All 21 test strains except for S. maltophilia were negative for bla L1, indicative of the high-specificity of the primers for the bla L1. A total of 22 L1-positive isolates were identified for LAMP-based surveillance of bla L1 from 105 ICU patients with clinically suspected multi-resistant infections. The sequences of these bla L1 genes were conservative with only a few sites mutated, and the strains had highly resistant to \u03b2-lactam antibiotics. The MLST recovered that 22 strains belonged to seven different S. maltophilia sequence types (STs). Furthermore, co-occurrence of bla L1 and bla L2 genes were detected in all of isolates. Strikingly, S. maltophilia DCPS-01 was recovered to contain bla L1, bla L2, and bla NDM-1 genes, possessing an ability to hydrolyse all \u03b2-lactams antibiotics. Our data showed the diversity types of S. maltophilia carrying bla L1 and co-occurrence of many resistant genes in the clinical strains signal an ongoing and fast evolution of S. maltophilia resulting from their wide spread in the respiratory infections, and therefore will be difficult to control.","lamp_id":[{"id":"LPB00593","pathogen":"Stenotrophomonas maltophilia","target":"blaL1"}]},{"id":324,"pmid":25529001,"title":"Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP)","year":2014,"journal":"Journal of Infection and Chemotherapy","authors":"Ryuichi Nakano, Akiyo Nakano, Yoshikazu Ishii, Tsuneyuki Ubagai, Takane Kikuchi-Ueda, Hirotoshi Kikuchi, Shigeru Tansho-Nagakawa, Go Kamoshida, Xiaoqin Mu, Yasuo Ono","doi":"10.1016\/j.jiac.2014.11.010","country":"Japan","institute":"Teikyo University School of Medicine","deparment":"Department of Microbiology and Immunology","abstract":"Klebsiella pneumoniae carbapenemases (KPC), which are associated with resistance to carbapenem, have recently spread worldwide and have become a global concern. It is necessary to detect KPC-producing organisms in clinical settings to be able to control the spread of this resistance. We have developed a loop-mediated isothermal amplification (LAMP) method for rapid detection of KPC producers. LAMP primer sets were designed to recognize the homologous regions of blaKPC-2 to blaKPC-17 and could amplify blaKPC rapidly. The specificity and sensitivity of the primers in the LAMP reactions for blaKPC detection were determined. This LAMP assay was able to specifically detect KPC producers at 68 \u00b0C, and no cross-reactivity was observed for other types of \u03b2-lactamase (class A, B, C, or D) producers. The detection limit for this assay was found to be 10(0) CFU per tube, in 25 min, which was 10-fold more sensitive than a PCR assay for blaKPC detection. Then, the sensitivity of the LAMP reactions for blaKPC detection in human specimens (sputum samples, urine samples, fecal samples and blood samples) was analyzed; it was observed that the LAMP assay had almost the same sensitivity in these samples as when using purified DNA. The LAMP assay is easy to perform and rapid. It may therefore be routinely applied for detection of KPC producers in the clinical laboratory.","lamp_id":[{"id":"LPB00594","pathogen":"Klebsiella pneumoniae","target":"KPC"}]},{"id":325,"pmid":25455747,"title":"Loop-mediated isothermal amplification with the Procedure for Ultra Rapid Extraction kit for the diagnosis of pneumocystis pneumonia","year":2014,"journal":"Journal of Infection and Chemotherapy","authors":"Shuichi Kawano, Takuya Maeda, Takefumi Suzuki, Tatsuhiro Abe, Kei Mikita, Yusuke Hamakawa, Takeshi Ono, Wataru Sonehara, Yasushi Miyahira, Akihiko Kawana","doi":"10.1016\/j.jiac.2014.09.004","country":"Japan","institute":"Department of Internal Medicine","deparment":"Division of Infectious Diseases and Pulmonary Medicine","abstract":"Loop-mediated isothermal amplification (LAMP) is an innovative molecular technique requiring only a heating device and isothermal conditions to amplify a specific target gene. The results of current microscopic diagnostic tools for pneumocystis pneumonia are not sufficiently consistent for detecting infection with a low-density of Pneumocystis jirovecii. Although polymerase chain reaction (PCR) is highly sensitive, it is not suitable for resource-limited facilities. LAMP is a potential diagnostic replacement for PCR in such settings but a critical disadvantage of DNA extraction was still remained. Therefore, we employed the Procedure for Ultra Rapid Extraction (PURE) kit, which uses a porous material, to isolate the DNA from clinical samples in a simple way in combination with previously reported LAMP procedure for diagnosing PCP. The detection limit of the PURE-LAMP method applied to artificial bronchoalveolar lavage fluid samples was 100 copies\/tube, even with the use of massive blood-contaminated solutions. In addition, we concluded the diagnostic procedure within 1 h without the need for additional equipment. PURE-LAMP coupled with suitable primers for specific pathogens has good potential for diagnosing various infectious diseases.","lamp_id":[{"id":"LPB00595","pathogen":"Pneumocystis jirovecii","target":"18S rRNA"}]},{"id":326,"pmid":25349088,"title":"Rapid detection of Staphylococcus aureus by loop-mediated isothermal amplification","year":2014,"journal":"Applied Biochemistry and Biotechnology","authors":"Xin-Ru Wang, Li-Fen Wu, Yan Wang, Ying-Ying Ma, Feng-Hua Chen, Hong-Ling Ou","doi":"10.1007\/s12010-014-1328-x","country":"","institute":"","deparment":"","abstract":"Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is a major bacterial pathogen associated with nosocomial and community-acquired S. aureus infections all over the world. A rapid detection assay for staphylococcal gene of nuc and mecA is needed. In this study, a rapid identification assay based on the loop-mediated isothermal amplification (LAMP) method was established. PCR and LAMP assays were used to detect Staphylococcus aureus and other related species for nuc and mecA. With optimization of the primers and reaction temperature, the LAMP successfully amplified the genes under isothermal conditions at 62 \u00b0C within 60 min, of which the results were identical with those of the conventional PCR methods. The detection limits of the LAMP for nuc and mecA were 1.47 and 14.7 pg\/\u03bcl DNA per tube, respectively, by naked eye inspections, while the detection limits of the PCR for nuc and mecA were 14.7 pg\/\u03bcl and 147 pg\/\u03bcl DNA, respectively. Finally, The LAMP method was then applied to clinical blood plaque samples. The LAMP and PCR demonstrated identical results for the plaque samples with the culture assay. Together, the LAMP offers an alternative detection assay for nuc and mecA with a great advantage of the sensitivity and rapidity.","lamp_id":[{"id":"LPB00596","pathogen":"Staphylococcus aureus","target":"nuc"},{"id":"LPB00733","pathogen":"Staphylococcus aureus","target":"mecA"}]},{"id":327,"pmid":25276360,"title":"Rapid detection of Streptococcus pneumoniae by real-time fluorescence loop-mediated isothermal amplification","year":2014,"journal":"Journal of Thoracic Disease","authors":"Yong Xia, Xu-Guang Guo, Shan Zhou","doi":"10.3978\/j.issn.2072-1439.2014.07.29","country":"China","institute":"The Third Clinical College of Guangzhou Medical University","deparment":"Department of Clinical Laboratory Medicine\/Department of Internal Medicine\/Center for Severe maternal Treatment of Guangzhou City\/Center for Clinical Laboratory Medicine of PLA","abstract":"Background and aim of study: A significant human pathogenic bacterium, Streptococcus pneumoniae was recognized as a major cause of pneumonia, and is the subject of many humoral immunity studies. Diagnosis is generally made based on clinical suspicion along with a positive culture from a sample from virtually any place in the body. But the testing time is too long. This study is to establish a rapid diagnostic method to identification of Streptococcus pneumoniae.\n\nMethods: Our laboratory has recently developed a new platform called real-amp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of Streptococcus pneumonia. Two pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the Streptococcus pneumoniae. The amplification was carried out at 63 degree Celsius using SYBR Green for 60 minutes with the tube scanner set to collect fluorescence signals. Clinical samples of Streptococcus pneumoniae and other bacteria were used to determine the sensitivity and specificity of the primers by comparing with traditional culture method.\n\nResults: The new set of primers consistently detected in laboratory-maintained isolates of Streptococcus pneumoniae from our hospital. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting Streptococcus pneumoniae.\n\nConclusions: This study demonstrates that the Streptococcus pneumoniae LAMP primers developed here have the ability to accurately detect Streptococcus pneumoniae infections by real-time fluorescence LAMP.","lamp_id":[{"id":"LPB00597","pathogen":"Streptococcus pneumoniae","target":"ply"}]},{"id":328,"pmid":25218428,"title":"Loop-mediated isothermal amplification assay for rapid and sensitive diagnosis of tuberculosis","year":2014,"journal":"Journal of Infection","authors":"Parveen Kumar, Deepal Pandya, Niti Singh, Digambar Behera, Praveen Aggarwal, Sarman Singh","doi":"10.1016\/j.jinf.2014.08.017","country":"India","institute":"All India Institute of Medical Sciences","deparment":"Division of Clinical Microbiology and Molecular Medicine","abstract":"Objectives: Loop-mediated isothermal amplification (LAMP) is a newly developed molecular method that can be performed isothermally. We developed and evaluated a LAMP assay using novel primers to diagnose tuberculosis directly from clinical samples.\n\nMaterials: Primers were designed to amplify the specific novel esat-6 gene target of Mycobacterium tuberculosis (MTB). Quantitated DNA was used to determine analytical sensitivity and specificity was evaluated by testing 29 NTM and 37 other bacterial species. After standardization, its sensitivity and specificity were evaluated on samples from 118 TB suspected and 31 non-TB patients and compared it with smear, culture and mPCR methods.\n\nResults: LAMP was able to detect 5 fg DNA (one MTB) within 21 min and found to be 10 times more sensitive than mPCR and showed 100% specificity against NTM and other bacterial species. In clinical samples, LAMP showed highest MTB detection rate (52.5%) as compared to mPCR (44%) and culture (30.5%). On culture positive and mPCR positive samples, the sensitivity of LAMP was found to be 100% (95% CI 90.2-100) and 96.1% (95% CI 86.7-99.5) respectively with 93.5% (95% CI 78.5-99.2) of overall specificity.\n\nConclusion: LAMP was found to be more sensitive than culture and mPCR for the detection of MTB. It showed specificity comparable to mPCR but was rapid and cost effective.","lamp_id":[{"id":"LPB00598","pathogen":"Mycobacterium tuberculosis","target":"esat6"}]},{"id":329,"pmid":24937215,"title":"Direct detection of Mycobacterium avium in environmental water and scale samples by loop-mediated isothermal amplification","year":2014,"journal":"Journal of Water and Health","authors":"Yukiko Nishiuchi, Aki Tamaru, Yasuhiko Suzuki, Seigo Kitada, Ryoji Maekura, Yoshitaka Tateishi, Mamiko Niki, Hisashi Ogura, Sohkichi Matsumoto","doi":"10.2166\/wh.2013.007","country":"Japan","institute":"Osaka City University Medical School","deparment":"Toneyama Institute for Tuberculosis Research","abstract":"We previously demonstrated the colonization of Mycobacterium avium complex in bathrooms by the conventional culture method. In the present study, we aimed to directly detect M. avium organisms in the environment using loop-mediated isothermal amplification (LAMP), and to demonstrate the efficacy of LAMP by comparing the results with those obtained by culture. Our data showed that LAMP analysis has detection limits of 100 fg DNA\/reaction for M. avium. Using an FTA(\u00ae) elute card, DNA templates were extracted from environmental samples from bathrooms in the residences of 29 patients with pulmonary M. avium disease. Of the 162 environmental samples examined, 143 (88%) showed identical results by both methods; 20 (12%) and 123 (76%) samples were positive and negative, respectively, for M. avium. Of the remaining 19 samples (12%), seven (5%) and 12 (7%) samples were positive by the LAMP and culture methods, respectively. All samples that contained over 20 colony forming units\/primary isolation plate, as measured by the culture method, were also positive by the LAMP method. Our data demonstrate that the combination of the FTA elute card and LAMP can facilitate prompt detection of M. avium in the environment.","lamp_id":[{"id":"LPB00599","pathogen":"Mycobacterium avium 104","target":"16S rRNA"}]},{"id":330,"pmid":24274610,"title":"Isothermal DNA amplification coupled to Au-nanoprobes for detection of mutations associated to Rifampicin resistance in Mycobacterium tuberculosis","year":2013,"journal":"Journal of Nanobiotechnology","authors":"Bruno Veigas, Pedro Pedrosa, Isabel Couto, Miguel Viveiros, Pedro V Baptista","doi":"10.1186\/1477-3155-11-38","country":"Portugal","institute":"Universidade Nova de Lisboa","deparment":"Departamento de Ci\u00eancias da Vida","abstract":"Background: Tuberculosis accounted for 8.7 million new cases in 2011 and continues to be one of the leading human infectious diseases. Burdensome is the increasing rate of multi-drug resistant tuberculosis (MDRTB) and the difficulties created for treatment and public health control programs, especially in developing countries. Resistance to rifampicin (RIF), a first line antibiotic, is commonly associated with point mutations within the rpoB gene of Mycobacterium tuberculosis (Mtb) whose detection is considered the best early molecular predictor for MDRTB. Gold nanoparticles functionalized with thiol-modified oligonucleotides (Au-nanoprobes) have shown the potential to provide a rapid and sensitive detection method for Mtb and single base alterations associated with antibiotic resistance, namely in rpoB gene associated to RIF resistance.\n\nResults: We developed a strategy based on the isothermal amplification of sample DNA (LAMP) coupled to specific Au-nanoprobes capable of identifying members of the Mtb complex (MTBC) and discriminating specific mutations within the rpoB gene. Integration of LAMP and Au-nanoprobe assay allowed to detect MTBC member and identify mutations linked to RIF resistance. A total of 12 biological samples were tested and a 100% specificity and sensitivity was attained.\n\nConclusions: There is an increasing demand for simple, fast and cheap methods for the molecular identification of Mtb and for the detection of molecular tags associated to drug resistance suitable for use at point-of-need. Here we describe such a method, that as the potential to get molecular diagnostic of tuberculosis to remote environments.","lamp_id":[{"id":"LPB00600","pathogen":"Mycobacterium tuberculosis H37Rv","target":"rpoB"}]},{"id":331,"pmid":23696915,"title":"Rapid, simple and sensitive detection of Q fever by loop-mediated isothermal amplification of the htpAB gene","year":2013,"journal":"PLOS Neglected Tropical Diseases","authors":"Lei Pan, Lijuan Zhang, Desheng Fan, Xiuchun Zhang, Hong Liu, Qunying Lu, Qiyi Xu","doi":"10.1371\/journal.pntd.0002231","country":"China","institute":"National Institute of Communicable Disease Control and Prevention","deparment":"Dept. of Rickettsiology","abstract":"Background: Q fever is the most widespread zoonosis, and domestic animals are the most common sources of transmission. It is not only difficult to distinguish from other febrile diseases because of the lack of specific clinical manifestations in humans, but it is also difficult to identify the disease in C. burnetii-carrying animals because of the lack of identifiable features. Conventional serodiagnosis requires sera from the acute and convalescent stages of infection, which are unavailable at early diagnosis. Nested PCR and real-time PCR require equipment. In this study, we developed a Loop-Mediated Isothermal Amplification (LAMP) assay to identify C. burnetii rapidly and sensitively.\n\nMethods: A universal LAMP primer set was designed to detect the repeated sequence IS1111a of the htpAB gene of C. burnetii using PrimerExplorer V4 software. The sensitivity of the LAMP assay was evaluated using known quantities of recombined reference plasmids containing the targeted genes. The specificity of the developed LAMP assay was determined using 26 members of order Rickettsiae and 18 other common pathogens. The utility of the LAMP assay was further compared with real time PCR by the examination 24 blood samples including 6 confirmed and 18 probable Q fever cases, which diagnosed by IFA serological assessment and real time PCR. In addition, 126 animal samples from 4 provinces including 97 goats, 7 cattle, 18 horses, 3 marmots and 1 deer were compared by these two methods.\n\nResults: The limits of detection of the LAMP assay for the htpAB gene were 1 copy per reaction. The specificity of the LAMP assay was 100%, and no cross-reaction was observed among the bacteria used in the study. The positive rate of unknown febrile patients was 33.3%(95%CI 30.2%-36.4%) for the LAMP assay and 8.3%(95%CI 7.4%-9.2%) for the real time PCR(P<0.05). Similarly, the total positive rate of animals was 7.9%(95%CI 7.1%-8.7%) for the LAMP assay and 0.8%(95%CI 0.7%-0.9%)for the real time PCR(P<0.01). Using the developed LAMP assay, Q fever in the Yi Li area, Xinjiang Province, was confirmed.\n\nConclusions: The LAMP assay is a potential tool to support the diagnosis of Q fever in humans and domestic animals in the field, especially in the rural areas of China, because of its rapid and sensitive detection without the aid of sophisticated equipment or a complicated protocol.","lamp_id":[{"id":"LPB00601","pathogen":"Coxiella burnetii RSA 493","target":"htpAB"}]},{"id":332,"pmid":23555102,"title":"Detection of Mycobacterium tuberculosis by using loop-mediated isothermal amplification combined with a lateral flow dipstick in clinical samples","year":2013,"journal":"BioMed Research International","authors":"Thongchai Kaewphinit, Narong Arunrut, Wansika Kiatpathomchai, Somchai Santiwatanakul, Pornpun Jaratsing, Kosum Chansiri","doi":"10.1155\/2013\/926230","country":"Thailand","institute":"Srinakharinwirot University","deparment":"Innovative Learning Center","abstract":"Tuberculosis (TB) is a communicable disease caused by the bacterium Mycobacterium tuberculosis (MTB) and is a persistent problem in the developing countries. Loop-mediated isothermal amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. Here, a LAMP method was combined with a chromatographic lateral-flow dipstick (LFD) to detect IS6110 gene of M. tuberculosis specifically and rapidly. The reaction was optimized at 63\u00b0C for 60 min, and the amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5 min was detected at the LFD test line 5 min after application. Excluding the step of DNA extraction, the test results could be generated approximately within 1 h. In addition to the advantage of short assay time, this technique could avoid the contact of carcinogenic ethidium bromide due to the exclusion of the electrophoresis analysis step. Furthermore, the data indicated that LAMP-LFD could detect M. tuberculosis genomic DNA as little as 5 pg. The technique showed a significant specificity since no cross-hybridization to M. intracellulare (MIC), M. fortuitum (MFT), M. avium (MAV), M. kansasii (MKS), and M. gordonae (MGD) genomic DNAs was observed. In the clinical unknown samples test, the sensitivity of LAMP-LFD was 98.92 % and the specificity was 100 % compared to those of the standard culture assay. Based on its sensitivity, specificity, rapidity, low cost, and convenience, LAMP-LFD could be applicable for use in both laboratories and epidemiological surveys of MTB.","lamp_id":[{"id":"LPB00602","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"}]},{"id":333,"pmid":23466595,"title":"Clinical value of IS6110-based loop-mediated isothermal amplification for detection of Mycobacterium tuberculosis complex in respiratory specimens","year":2013,"journal":"Journal of Infection","authors":"Ehsan Aryan, Manoochehr Makvandi, Ahmad Farajzadeh, Kris Huygen, Amir-Hooshang Alvandi, Mohammad-Mehdi Gouya, Ali Sadrizadeh, Marta Romano","doi":"10.1016\/j.jinf.2013.02.005","country":"Iran","institute":"Mashhad University of Medical Sciences","deparment":"Department of Medical Microbiology","abstract":"Objectives: A fundamental to global tuberculosis (TB) control is timely and accurate diagnosis of infectious cases of the disease. Among various methods, techniques based on nucleic acid amplification are the ones with promising prospects. The present study evaluates the diagnostic value of the recently developed IS6110-based loop-mediated isothermal amplification (LAMP) for detection of Mycobacterium tuberculosis complex (MTBC) in sputum specimens.\n\nMethods: In this cross-sectional study (2008-2009), IS6110-LAMP was evaluated on 101 sputum specimens from 93 highly suspected TB patients and compared to Amplicor MTB test and in-house IS6110-PCR and -nested PCR assays. Culture results or clinical recovery following anti-TB therapy was considered as a reference to prove the TB cases.\n\nResults: The overall sensitivity of IS6110-LAMP, Amplicor, nPCR, and PCR were respectively 89.6% (69\/77 specimens; 95% confidence interval [CI], 80.5-95.4%), 76.6% (59\/77 specimens; CI, 65.6-85.5%), 79.2% (61\/77 specimens; CI, 68.5-87.6%) and 59.7% (46\/77 specimens; CI, 47.9-70.8%). The specificity and positive predictive value (PPV) were 100% for all the tests, and the negative predictive value (NPV) of IS6110-LAMP, Amplicor, nPCR, and PCR were respectively 75%, 57.1%, 60%, and 43.6%. There was an excellent overall agreement between LAMP and nPCR (k 0.828), and between LAMP and Amplicor (k 0.746), in addition to a better tolerance of IS6110-LAMP to inhibitors present in clinical specimens.\n\nConclusion: The better diagnostic performance of IS6110-LAMP compared to Amplicor (p = 0.009), nPCR (p = 0.013) and PCR (p < 0.0001) besides its rapidity, simplicity, and cost-effectiveness makes it a valuable method for the detection of MTBC in clinical samples, particularly in resource-limited settings.","lamp_id":[{"id":"LPB00603","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"}]},{"id":334,"pmid":35738204,"title":"One-step colorimetric isothermal detection of COVID-19 with AI-assisted automated result analysis: A platform model for future emerging point-of-care RNA\/DNA disease diagnosis","year":2022,"journal":"Talanta","authors":"Wansadaj Jaroenram, Itthi Chatnuntawech, Jantana Kampeera, Sukanya Pengpanich, Pornsawan Leaungwutiwong, Benyatip Tondee, Sarawut Sirithammajak Rapheephat Suvannakad, Pakapreud Khumwan, Sirintip Dangtip, Narong Arunrut, Sirasate Bantuchai, Wang Nguitragool, Suchawit Wongwaroran, Paisan Khanchaitit, Jetsumon Sattabongkot, Surat Teerapittayanon, Wansika Kiatpathomchai","doi":"10.1016\/j.talanta.2022.123375","country":"","institute":"","deparment":"","abstract":"Colorimetric loop-mediated DNA isothermal amplification-based assays have gained momentum in the diagnosis of COVID-19 owing to their unmatched feasibility in low-resource settings. However, the vast majority of them are restricted to proprietary pH-sensitive dyes that limit downstream assay optimization or hinder efficient result interpretation. To address this problem, we developed a novel dual colorimetric RT-LAMP assay using in-house pH-dependent indicators to maximize the visual detection and assay simplicity, and further integrated it with the artificial intelligence (AI) operated tool (RT-LAMP-DETR) to enable a more precise and rapid result analysis in large scale testing. The dual assay leverages xylenol orange (XO) and a newly formulated lavender green (LG) dye for distinctive colorimetric readouts, which enhance the test accuracy when performed and analyzed simultaneously. Our RT-LAMP assay has a detection limit of 50 viral copies\/reaction with the cycle threshold (Ct) value \u2264 39.7 \u00b1 0.4 determined by the WHO-approved RT-qPCR assay. RT-LAMP-DETR exhibited a complete concordance with the results from naked-eye observation and RT-qPCR, achieving 100% sensitivity, specificity, and accuracy that altogether render it suitable for ultrasensitive point-of-care COVID-19 screening efforts. From the perspective of pandemic preparedness, our method offers a simpler, faster, and cheaper (\u223c$8\/test) approach for COVID-19 testing and other emerging pathogens with respect to RT-qPCR.","lamp_id":[{"id":"LPB00604","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP9)"}]},{"id":335,"pmid":22627308,"title":"A rapid loop-mediated isothermal amplification assay targeting hspX for the detection of Mycobacterium tuberculosis complex","year":2012,"journal":"Japanese Journal of Infectious Diseases","authors":"Aixiao Bi, Chie Nakajima, Yukari Fukushima, Aki Tamaru, Isamu Sugawara, Akio Kimura, Ryuji Kawahara, Zhongyi Hu, Yasuhiko Suzuki","doi":"10.7883\/yoken.65.247","country":"China","institute":"Tongji University School of Medicine","deparment":"Shanghai Key Laboratory of Tuberculosis","abstract":"A rapid, simple, and low-cost diagnostic tool for tuberculosis (TB) detection is urgently needed in countries with a high TB burden. Here, we report a novel loop-mediated isothermal amplification (LAMP) assay targeting the hspX gene for the rapid detection of Mycobacterium tuberculosis, M. bovis, M. africanum, and M. microti. The specificity of this assay was evaluated using 4 reference strains of Mycobacterium tuberculosis complex (MTC), 22 species of non-tuberculous mycobacteria (NTM), 7 non-mycobacterial species, and 50 clinical M. tuberculosis isolates. All the reference MTC strains and M. tuberculosis clinical isolates were successfully detected by this method, and there were no false-positive results with NTM or non-mycobacterial species, which demonstrates the high specificity of this assay for MTC. The detection limit was 10 copies of MTC genome within 27 min, and the detection speed of this assay was higher than that of any other isothermal methods reported so far. Because of its speed, simplicity, sensitivity, specificity, and inexpensiveness, the TB hspX LAMP assay is a potential gene diagnostic method for TB detection in developing countries with a high TB burden.","lamp_id":[{"id":"LPB00605","pathogen":"Mycobacterium tuberculosis H37Rv","target":"hspX"}]},{"id":336,"pmid":22806847,"title":"A modified visual loop-mediated isothermal amplification method for diagnosis and differentiation of main pathogens from Mycobacterium tuberculosis complex","year":2012,"journal":"World Journal of Microbiology and Biotechnology","authors":"Ming Hong, Lei Zha, Wenliang Fu, Minji Zou, Wuju Li, Donggang Xu","doi":"10.1007\/s11274-011-0843-y","country":"China","institute":"Beijing Institute of Basic Medical Sciences","deparment":"Department of Genome Engineering","abstract":"This study was aimed to rapidly identify and differentiate two main pathogens of the Mycobacterium tuberculosis complex: Mycobacterium tuberculosis subsp. tuberculosis and Mycobacterium bovis by a modified loop-mediated isothermal amplification (LAMP) assay. The reaction results could be evaluated by naked eye with two optimized closed tube detection methods as follows: adding the modified fluorescence dye in advance into the reaction mix so as to observe the color changes or putting a tinfoil in the tube and adding the SYBR Green I dye on it, then making the dye drop into the bottom of the tube by centrifuge after reaction. The results showed that the two groups of primers used jointly in this assay could successfully identify and differentiate Mycobacterium tuberculosis subsp. tuberculosis and Mycobacterium tuberculosis bovis. Sensitivity test displayed that the modified LAMP assay with the closed tube system could determine the minimal template concentration of 1 copy\/\u03bcl, which was more sensitive than that of routine PCR. The advantages of this LAMP method for detection of the Mycobacterium tuberculosis complex included high specificity, high sensitivity, simplicity, and superiority in avoidance of aerosol contamination. The modified LAMP assay would provide a potential for clinical diagnosis and therapy of tuberculosis in the developing countries and the resource-limited areas.","lamp_id":[{"id":"LPB00606","pathogen":"Mycobacterium tuberculosis H37Rv","target":"esat6"}]},{"id":337,"pmid":30105321,"title":"Multiplex sample-to-answer detection of bacteria using a pipette-actuated capillary array comb with integrated DNA extraction, isothermal amplification, and smartphone detection","year":2018,"journal":"Lab on a Chip","authors":"Junhou Hui, Yin Gu, Yuanshou Zhu, Yanjing Chen, Shu-Juan Guo, Sheng-Ce Tao, Yan Zhang, Peng Liu","doi":"10.1039\/c8lc00543e","country":"","institute":"","deparment":"","abstract":"A pipette-actuated capillary array comb (PAAC) system operated on a smartphone-based hand-held device has been successfully developed for the multiplex detection of bacteria in a \"sample-to-answer\" manner. The PAAC consists of eight open capillaries inserted into a cylindrical plastic base with a piece of chitosan-modified glass filter paper embedded in each capillary. During the sample preparation, a PAAC was mounted into a 1 mL pipette tip with an enlarged opening and was operated with a 1 mL pipette for liquid handling. The cell lysate was drawn and expelled through the capillaries three times to facilitate the DNA capture on the embedded filter discs. Following washes with water, the loop-mediated isothermal amplification (LAMP) reagents were aspirated into the capillaries, in which the primers were pre-fixed with chitosan. After that, the PAAC was loaded into the smartphone-based device for a one-hour amplification at 65 \u00b0C and end-point detection of calcein fluorescence in the capillaries. The DNA capture efficiency of a 1.1 mm-diameter filter disc was determined to be 97% of \u03bb-DNA and the coefficient of variation among the eight capillaries in the PAAC was only 2.2%. The multiplex detection of genomic DNA extracted from Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus provided limits of detection of 200, 500, and 500 copies, respectively, without any cross-contamination and cross reactions. \"Sample-to-answer\" detection of E. coli samples was successfully completed in 85 minutes, demonstrating a sensitivity of 200 cfu per capillary. The multiplex \"sample-to-answer\" detection, the streamlined operation, and the compact device should facilitate a broad range of applications of our PAAC system in point-of-care testing.","lamp_id":[{"id":"LPB00607","pathogen":"Staphylococcus aureus","target":null},{"id":"LPB00754","pathogen":"Klebsiella pneumoniae","target":null}]},{"id":338,"pmid":20673837,"title":"One-tube loop-mediated isothermal amplification combined with restriction endonuclease digestion and ELISA for colorimetric detection of resistance to isoniazid, ethambutol and streptomycin in Mycobacterium tuberculosis isolates","year":2010,"journal":"Journal of Microbiological Methods","authors":"Mei-Feng Lee, Yen-Hsu Chen, Hui-Jine Hsu, Chien-Fang Peng","doi":"10.1016\/j.mimet.2010.07.018","country":"Taiwan","institute":"Kaohsiung Medical University","deparment":"Department of Medical Laboratory Science and Biotechnology","abstract":"In this study, we designed a simple and rapid colorimetric detection method, a one-tube loop-mediated isothermal amplification (LAMP)-PCR-hybridization-restriction endonuclease-ELISA [one-tube LAMP-PCR-HY-RE-ELISA] system, to detect resistance to isoniazid, ethambutol and streptomycin in strains of Mycobacterium tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting isoniazid-resistant, ethambutol-resistant and streptomycin-resistant isolates of M. tuberculosis showed 98.9%, 94.3% and 93.8%, respectively. This assay is rapid and convenient that can be performed within one working day. One-tube LAMP-PCR-HY-RE-ELISA system was designed based on hot spot point mutations in target drug-resistant genes, using LAMP-PCR, hybridization, digestion with restriction endonuclease and colorimetric method of ELISA. In this study, LAMP assay was used to amplify DNA from drug-resistant M. tuberculosis, and ELISA was used for colorimetrical determination. This assay will be a useful tool for rapid diagnosis of mutant codons in strains of M. tuberculosis for isoniazid at katG 315 and katG 463, ethambutol at embB 306 and embB 497, and streptomycin at rpsL 43.","lamp_id":[{"id":"LPB00608","pathogen":"Mycobacterium tuberculosis H37Rv","target":"katG"},{"id":"LPB00609","pathogen":"Mycobacterium tuberculosis H37Rv","target":"katG"},{"id":"LPB00610","pathogen":"Mycobacterium tuberculosis H37Rv","target":"embB"},{"id":"LPB00611","pathogen":"Mycobacterium tuberculosis H37Rv","target":"embB"},{"id":"LPB00612","pathogen":"Mycobacterium tuberculosis H37Rv","target":"rpsL"}]},{"id":339,"pmid":26338293,"title":"Detection of Mycobacterium tuberculosis complex in sputum specimens using a loop-mediated isothermal amplification assay in Korea","year":2015,"journal":"Journal of Medical Microbiology","authors":"Se Hoon Moon, Eun Jin Kim, Jun Tomono, Shigehiko Miyamoto, Satoshi Mitarai, Dong Wook Kim, Mitsuko Seki","doi":"10.1099\/jmm.0.000164","country":"Republic of Korea","institute":"Hanyang University","deparment":"Department of Pharmacy","abstract":"Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis complex (MTC), remains one of the leading causes of death in the world. In Korea, the current prevalence of multidrug-resistant TB (MDR-TB) poses a major problem. The most common method for diagnosing TB in developing countries is sputum smear microscopy; however, the sensitivity of this test is relatively low and it usually requires well-trained laboratory staff. Cultures of MTC require up to several weeks in sophisticated facilities, such as Biosafety Level 3. Effective diagnostic techniques are necessary to control TB. In Korea, we evaluated a loop-mediated isothermal amplification (LAMP) assay targeting the hspX gene (TB-hspX-LAMP) of MTC. For clinical evaluation, culture confirmation, smear microscopy and TB-hspX-LAMP were performed on 303 sputum specimens obtained from suspected TB patients in Korea. The sensitivity, specificity, positive predictive value and negative predictive value of TB-hspX-LAMP were 71.1, 98.8, 91.4 and 95.1%, respectively, compared with TB culture, which is the gold standard for diagnosis of TB. In contrast, the comparable values of smear microscopy were 24.4, 98.1, 68.8 and 88.2%, respectively. Therefore, we concluded that TB-hspX-LAMP was superior to the use of smear microscopy for the detection of MTC in sputum specimens in clinical settings in Korea.","lamp_id":[{"id":"LPB00613","pathogen":"Mycobacterium tuberculosis H37Rv","target":"hspX"}]},{"id":340,"pmid":26491278,"title":"Investigation of Pneumocystis jirovecii colonization in patients with chronic pulmonary diseases in the People's Republic of China","year":2015,"journal":"International Journal of Chronic Obstructive Pulmonary Disease","authors":"Dong-Dong Wang, Ming-Quan Zheng, Nan Zhang, Chun-Li An","doi":"10.2147\/COPD.S89666","country":"P. R. China","institute":"China Medical University","deparment":"Department of Obstetrics and Gynecology","abstract":"Background: The detection of Pneumocystis jirovecii DNA in respiratory specimen from individuals who do not have signs or symptoms of pneumonia has been defined as colonization. The role of P. jirovecii colonization in the development or progression of various lung diseases has been reported, but little information about P. jirovecii colonization in patients is available in the People's Republic of China.\n\nObjective: To determine the prevalence of P. jirovecii colonization in patients with various pulmonary diseases, including the acute and stable stage of COPD, interstitial lung diseases, cystic fibrosis, and chronic bronchiectasis.\n\nMaterials and methods: A loop-mediated isothermal amplification (LAMP) and a conventional polymerase chain reaction (PCR) method for detecting P. jirovecii were developed. Ninety-eight HIV-negative patients who were followed-up and who had undergone bronchoscopy for diagnosis of various underlying respiratory diseases were included in the study. Sputa of these patients were analyzed with LAMP amplification of P. jirovecii gene. In addition, conventional PCR, Giemsa and Gomori's methenamine silver nitrate staining assays were applied to all specimens.\n\nResults: The sensitivity and specificity test showed that there was no cross-reaction with other fungi or bacteria in detecting the specific gene of P. jirovecii by LAMP, and the minimum detection limits by LAMP was 50 copies\/mL. P. jirovecii DNA was detected in 62 of 98 (63.3%) sputa specimens by LAMP assay and 22.45% (22\/98) by conventional PCR. However, no P. jirovecii cysts were found by Giemsa and Gomori's methenamine silver nitrate in all of gene-positive specimens.\n\nConclusion: The results of our study showed that prevalence of P. jirovecii colonization is particularly high in patients with chronic pulmonary diseases in the People's Republic of China, and the LAMP method is better for evaluation of the colonization of P. jirovecii in sputum specimen than conventional PCR.","lamp_id":[{"id":"LPB00614","pathogen":"Pneumocystis jirovecii","target":"16S rRNA"}]},{"id":341,"pmid":26319025,"title":"Detection of Streptococcus pyogenes using rapid visual molecular assay","year":2015,"journal":"FEMS Microbiology Letters","authors":"Xiangna Zhao, Xiaoming He, Huan Li, Jiangtao Zhao, Simo Huang, Wei Liu, Xiao Wei, Yiwei Ding, Zhaoyan Wang, Dayang Zou, Xuesong Wang, Derong Dong, Zhan Yang, Xiabei Yan, Liuyu Huang, Shuangkui Du, Jing Yuan","doi":"10.1093\/femsle\/fnv148","country":"China","institute":"Academy of Military Medical Sciences","deparment":"Institute of Disease Control and Prevention","abstract":"Streptococcus pyogenes is an increasingly important pathogen in many parts of the world. Rapid and accurate detection of S. pyogenes aids in the control of the infection. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of S. pyogenes. The assay incorporates two methods: a chromogenic analysis using a calcein\/Mn(2+) complex and real-time turbidity monitoring to assess the reaction. Both methods detected the target DNA within 60 min under 64\u00b0C isothermal conditions. The assay used specifically designed primers to target spy1258, and correctly identified 111 strains of S. pyogenes and 32 non-S. pyogenes strains, including other species of the genus Streptococcus. Tests using reference strains showed that the LAMP assay was highly specific. The sensitivity of the assay, with a detection limit of 1.49 pg DNA, was 10-fold greater than that of PCR. The LAMP assay established in this study is simple, fast and sensitive, and does not rely upon any special equipment; thus, it could be employed in clinical diagnosis.","lamp_id":[{"id":"LPB00615","pathogen":"Streptococcus pyogenes M1 GAS","target":"spy1258"}]},{"id":342,"pmid":26554941,"title":"Selection of fluorescent DNA dyes for real-time LAMP with portable and simple optics","year":2015,"journal":"Journal of Microbiological Methods","authors":"Gregoire Seyrig, Robert D Stedtfeld, Dieter M Tourlousse, Farhan Ahmad, Keara Towery, Alison M Cupples, James M Tiedje, Syed A Hashsham","doi":"10.1016\/j.mimet.2015.11.004","country":"USA","institute":"Michigan State University","deparment":"Department of Civil and Environmental Engineering","abstract":"Loop-mediated isothermal amplification (LAMP) is increasingly used for point-of-care nucleic acid based diagnostics. LAMP can be monitored in real-time by measuring the increase in fluorescence of DNA binding dyes. However, there is little information comparing the effect of various fluorescent dyes on signal to noise ratio (SNR) or threshold time (Tt). This information is critical for implementation with field deployable diagnostic tools that require small, low power consumption, robust, and inexpensive optical components with reagent saving low volume reactions. In this study, SNR and Tt during real-time LAMP was evaluated with eleven fluorescent dyes. Of all dyes tested, SYTO-82, SYTO-84, and SYTOX Orange resulted in the shortest Tt, and SYTO-81 had the widest range of working concentrations. The optimized protocol detected 10 genome copies of Mycobacterium tuberculosis in less than 10 min, 10 copies of Giardia intestinalis in ~20 min, and 10 copies of Staphylococcus aureus or Salmonella enterica in less than 15 min. Results demonstrate that reaction efficiency depends on both dye type and concentration and the selected polymerase. The optimized protocol was evaluated in the Gene-Z\u2122 device, a hand-held battery operated platform characterized via simple and low cost optics, and a multiple assay microfluidic chip with micron volume reaction wells. Compared to the more conventional intercalating dye (SYBR Green), reliable amplification was only observed in the Gene-Z\u2122 when using higher concentrations of SYTO-81.","lamp_id":[{"id":"LPB00616","pathogen":"Mycobacterium tuberculosis","target":"16S rRNA"}]},{"id":343,"pmid":27213686,"title":"Rapid and simple identification of Beijing genotype strain of Mycobacterium tuberculosis using a loop-mediated isothermal amplification assay","year":2016,"journal":"Medical Microbiology and Immunology","authors":"Yuhki Nagai, Yoshito Iwade, Manabu Nakano, Shigehiro Akachi, Takashi Kobayashi, Takamichi Nishinaka","doi":"10.1111\/1348-0421.12389","country":"Japan","institute":"Mie Prefecture Health and Environment Research Institute","deparment":"","abstract":"Beijing genotype strains of Mycobacterium tuberculosis are geographically widespread and pose a notorious public health problem, these strains causing outbreaks of multidrug-resistant tuberculosis (TB); some studies have reported an association with drug resistance. Because the prevalence of Beijing strain has a substantial impact on TB control programs, the availability of a rapid and reliable method for detecting these strains is important for epidemiological monitoring of their circulation. The main methods currently used to identify Beijing genotype strains are IS6110 DNA fingerprinting, spoligotyping and PCR to detect specific deletions such as region of difference (RD)207. More recently, multiplex PCR assay using a Beijing-specific single nucleotide polymorphism (SNP) has been developed for detecting Beijing lineage strains. However, these methods are time-consuming and technically demanding. In the present study, a loop-mediated isothermal amplification (LAMP) assay that allows specific identification of Beijing genotype strain was developed. This Beijing genotype strain-identifying LAMP assay was performed 214 clinical isolates and the results compared with those of conventional PCR that targeted RD207 and Rv0679c-targreting multiplex PCR for Beijing lineage identification. LAMP assay showed 100% sensitivity and specificity compared with RD207-PCR. Furthermore, the sensitivity and specificity were 99.3% and 100%, respectively, compared with Rv0679c-multiplex PCR. This LAMP assay could be used routinely in local laboratories to monitor the prevalence of the Beijing genotype strain and thereby used to help control the spread of these potentially highly virulent and drug resistant strains.","lamp_id":[{"id":"LPB00617","pathogen":"Mycobacterium tuberculosis H37Rv","target":"RD207"}]},{"id":344,"pmid":27270464,"title":"Loop-mediated isothermal amplification assay targeting the mpb70 gene for rapid differential detection of Mycobacterium bovis","year":2016,"journal":"Archives of Microbiology","authors":"Hui Zhang, Zhen Wang, Xudong Cao, Zhengrong Wang, Jinliang Sheng, Yong Wang, Jing Zhang, Zhiqiang Li, Xinli Gu, Chuangfu Chen","doi":"10.1007\/s00203-016-1232-6","country":"P. R. China","institute":"Shihezi University","deparment":"College of Animal Science and Technology","abstract":"Loop-mediated isothermal amplification (LAMP) is a highly sensitive, rapid, cost-effective nucleic acid amplification method. Tuberculosis (TB) is widely popular in the world and it is difficult to cure. The fundamental treatment is to clear the types of TB pathogens such as Mycobacterium bovis (M. bovis), Mycobacterium tuberculosis (M. tuberculosis). In order to detect and diagnose TB early, we constructed the differential diagnostic method of TB. In this study, we used LAMP for detection of M. bovis, based on amplification of the mpb70 gene which is a unique gene in M. bovis strain. The LAMP assay was able to detect only seven copies of the gene per reaction, whereas for the conventional PCR, it was 70 copies. The LAMP was evaluated for its specificity using six strains of five Mycobacterium species and 18 related non-Mycobacterium microorganism strains as controls. The target three Mycobacterium strains were all amplified, and no cross-reaction was found with 18 non-Mycobacterium microorganism strains. TB was detected by two methods, LAMP and conventional PCR (based on mpb70 gene); the positive rates of the two methods were 9.55 and 7.01 %, respectively. Our results indicate that the LAMP method should be a potential tool with high convenience, rapidity, sensitivity and specificity for the diagnosis of TB caused by M. bovis. Most importance is that the use of LAMP as diagnostic method in association with diagnostic tests based on mpb70 gene would allow the differentiation between M. bovis and other Mycobacterium in humans or animals. The LAMP method is actually in order to detect human TB, and it can be used for differential diagnosis in this paper.","lamp_id":[{"id":"LPB00618","pathogen":"Mycobacterium tuberculosis variant bovis","target":"mpb70"}]},{"id":345,"pmid":36282886,"title":"Fully Automated CRISPR-LAMP Platform for SARS-CoV-2 Delta and Omicron Variants","year":2022,"journal":"Analytical Chemistry","authors":"Tong Zhang, Wang Zhao, Xi Chen,\u2020 Xinlian Zhang, Jinhui Zhu, Shenwei Li, Chuanyong Wu, Zhengan Tian, Guodong Sui","doi":"10.1021\/acs.analchem.2c03607","country":"","institute":"","deparment":"","abstract":"Integrated clustered regularly interspaced short palindromic repeat (CRISPR)-loop-mediated amplification (LAMP) technology is of great importance in CRISPR-based diagnostic systems, which urgently needs to be developed to improve diagnostic accuracy. A labor-free, contamination-free, and fully automated droplet manipulation platform for the CRISPR-LAMP technology has not been developed before. Herein, we propose a fully automated CRISPR-LAMP platform, which can precisely manipulate the CRISPR-LAMP droplet and perform combined reactions with high sensitivity and specificity. SARS-CoV-2 Spike T478K, D614G, P681R, and P681H mutations, typical point mutations of B.1.617.2 (Delta) and Omicron variants, are monitored with this platform with a detection limit of 10^2 copies\/\u03bcL. Allele discrimination between the mutants and wild type is significant with the designed one\/two-mismatch CRISPR RNA (crRNA) at a limit of 10^2 copies\/\u03bcL. Chemically synthesized and modified crRNAs greatly increase the CRISPR-LAMP signal, which advance the wide application. Combined with the previously developed RdRp CRISPR-LAMP assay, clinical results showed that Spike T478K and P681H can discriminate the mutant type form the wild type with 70% (49.66-85.50%, 95% confidence interval) and 78% (57.27-90.62%, 95% confidence interval) sensitivity, respectively, and 100% specificity (51.68-100%, 95% confidence interval), and the RdRp target can detect SARS-CoV-2 strains with 85% sensitivity (65.39-95.14%, 95% confidence interval) and 100% specificity (51.68-100%, 95% confidence interval). We believe that this automatic digital microfluid (DMF) system can advance the integrated CRISPR-LAMP technology with higher stability, sensitivity, and practicability, also for other CRISPR-associated diagnostic platforms.","lamp_id":[{"id":"LPB00619","pathogen":"SARS-CoV-2","target":"RdRp"},{"id":"LPB00841","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00842","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00843","pathogen":"SARS-CoV-2","target":"S"}]},{"id":346,"pmid":27117517,"title":"Rapid detection of measles virus using reverse transcription loop-mediated isothermal amplification coupled with a disposable lateral flow device","year":2016,"journal":"Diagnostic Microbiology and Infectious Disease","authors":"Changping Xu, Yan Feng Yin Chen, Jian Gao, Yiyu Lu","doi":"10.1016\/j.diagmicrobio.2016.02.023","country":"P.R China","institute":"Zhejiang provincial Center for Disease Control and Prevention","deparment":"Key Lab. of emergency detection for Public Health of Zhejiang province","abstract":"The measles virus (MeV) causes a highly contagious disease and efforts to reduce its spread are critical. A reverse transcription loop-mediated isothermal amplification assay coupled with a disposable lateral flow device (RT-LAMP-LFD) was developed for the rapid detection of MeV. The assay was performed in 40 min at an optimal temperature of 58 \u00b0C, with endpoint results visualized directly. A probe that was complementary to the RT-LAMP amplicon was designed to enhance assay specificity. Detection limit of the assay was 8.8 copies\/\u03bcL synthetic RNA, which equals the sensitivity of real-time RT-PCR. Clinical specimens were used to validate the RT-LAMP-LFD in provincial Center for Disease Control and Prevention (CDC) (n = 245) and six municipal CDCs (n = 249). The results obtained using RT-LAMP-LFD and real-time RT-PCR were highly concordant. The RT-LAMP-LFD is rapid, stable, and does not require expensive equipment, which can be used for routine MeV monitoring in CDC laboratories.","lamp_id":[{"id":"LPB00620","pathogen":"Measles morbillivirus","target":"H"}]},{"id":347,"pmid":15814969,"title":"Loop-mediated isothermal amplification method targeting the lytA gene for detection of Streptococcus pneumoniae","year":2005,"journal":"Journal of Clinical Microbiology","authors":"Mitsuko Seki, Yoshihisa Yamashita, Hirotaka Torigoe, Hiromasa Tsuda, Setsuko Sato, Masao Maeno","doi":"10.1128\/JCM.43.4.1581-1586.2005","country":"Japan","institute":"Nihon University School of Dentistry","deparment":"Department of Oral Health Sciences","abstract":"It is difficult to separate Streptococcus pneumoniae from the genotypically similar species Streptococcus mitis and Streptococcus oralis, which are commensals of the human oral cavity. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63 degrees C) with high specificity, efficiency, and rapidity, was examined regarding its applicability for detecting S. pneumoniae. An S. pneumoniae-specific LAMP primer targeting the lytA gene was designed. The primer specificity was validated using 10 Streptococcus and 7 non-Streptococcus species. Within 60 min, the assay could detect 10 or more copies of purified S. pneumoniae DNA with a sensitivity 1,000 times that of conventional PCR. Clinical isolates of 21 other strains (3 S. oralis, 17 S. mitis, and 1 Streptococcus species) that harbor virulence-factor-encoding genes (lytA or ply) were tried to differentiate S. pneumoniae. The detection of S. pneumoniae in clinical isolates was more selective using the LAMP method than using conventional PCR. Therefore, LAMP appears to be a sensitive and reliable means of diagnosing S. pneumoniae infection.","lamp_id":[{"id":"LPB00621","pathogen":"Streptococcus pneumoniae R6","target":"lytA"}]},{"id":348,"pmid":25979593,"title":"An integrated microfluidic chip for the detection of bacteria - A proof of concept","year":2015,"journal":"Molecular and Cellular Probes","authors":"Zhe Guo, Ting Yu, Jiarui He, Fen Liu, Hualong Hao, Yang Zhao, Jiabin Wen, Qi Wang","doi":"10.1016\/j.mcp.2015.05.005","country":"","institute":"","deparment":"","abstract":"We designed a microfluidic chip as a proof of concept for the detection of bacterial DNA. The chip was fabricated with poly-dimethylsiloxane (PDMS). It included a solid phase extraction (SPE) chamber, two separate channels and multiple loop-mediated isothermal amplification (LAMP) chambers. Three bacterial strains (Escherichia coli O157:H7, methicillin-resistant Staphylococcus aureus and methicillin-sensitive S. aureus) were used to test the feasibility of the device. LAMP products were examined directly using a UV light and verified by agarose gel electrophoresis. Using this chip, we successfully detected E. coli O157:H7, MSSA and MRSA in less than 2 h. The detection limit for genes rfbE, spa and mecA (specific to E. coli O157:H7, MSSA and MRSA, respectively) was <10(2) CFU\/100 \u03bcl. Further work is required to refine this approach and rigorously assess its analytical and diagnostic specificity and sensitivity.","lamp_id":[{"id":"LPB00622","pathogen":"Staphylococcus aureus","target":"spa"},{"id":"LPB00833","pathogen":"Staphylococcus aureus","target":"mecA"}]},{"id":349,"pmid":15902708,"title":"A simple method for the detection of measles virus genome by loop-mediated isothermal amplification (LAMP)","year":2005,"journal":"Journal of Medical Virology","authors":"Motoko Fujino, Naoko Yoshida, Shinya Yamaguchi, Norimitsu Hosaka, Yoshinori Ota, Tsugunori Notomi, Tetsuo Nakayama","doi":"10.1002\/jmv.20371","country":"Japan","institute":"Kitasato Institute for Life Sciences","deparment":"Laboratory of Viral Infection I","abstract":"Approximately 20,000-30,000 measles patients were reported in a surveillance of infectious diseases because of low vaccine coverage of 80% in Japan. Among them, some were thought to be secondary vaccine failure (SVF) with generally mild or non-typical measles illness and sometimes became a source of further transmission. We have developed a new, sensitive, and rapid method to detect the measles virus genome by reverse transcription loop-mediated isothermal amplification (RT-LAMP). We examined 50 nasopharyngeal secretion (NPS) samples that were obtained during the 1999 outbreak and stored at -70 degrees C and fresh NPS, lymphocytes and sera from 11 patients in 2003. Total RNA was extracted from the samples and subjected to reverse transcription-polymerase chain reaction (RT-PCR) and RT-LAMP. We detected the genomic RNA corresponding to at least 0.01-0.04 TCID50, 30-100 copies in samples by RT-LAMP within 60 min after extraction of RNA, and all four genotypes isolated in Japan were equally amplified. Specific DNA amplification was monitored spectrophotometrically by real time turbidimeter and the quantity of RNA was calculated. Measles virus genome was detected in 44 of 50 stored NPS by RT-PCR and in 49 by RT-LAMP. The vaccine strain was discriminated from wild strains after sequencing the LAMP products. RT-LAMP is a useful rapid diagnostic method for the detection of measles virus without any special apparatus, showing higher sensitivity than RT-PCR, and expected to be applied for hospital-based infection control and for laboratory-based measles surveillance.","lamp_id":[{"id":"LPB00623","pathogen":"Measles morbillivirus","target":"N"}]},{"id":350,"pmid":27677540,"title":"Development and evaluation of an in-house single step loop-mediated isothermal amplification (SS-LAMP) assay for the detection of Mycobacterium tuberculosis complex in sputum samples from Moroccan patients","year":2016,"journal":"BMC Infectious Diseases","authors":"El Mehdi Bentaleb, Mohammed Abid, My Driss El Messaoudi, Brahim Lakssir, El Mostafa Ressami, Saa\u00efd Amzazi, Hassan Sefrioui, Hassan Ait Benhassou","doi":"10.1186\/s12879-016-1864-9","country":"Morocco","institute":"Innovation and Research (MAScIR)","deparment":"Moroccan Foundation for Advanced Science","abstract":"Background: Tuberculosis (TB) is a major global health problem and remains the leading cause of morbidity and mortality in developing countries. Routinely used TB diagnostic methods, in most endemic areas, are time-consuming, often less-sensitive, expensive and inaccessible to most patients. Therefore, there is an urgent need for the development of early, easy to use and effective diagnosis tools of TB, which can be effectively integrated into resource limited settings, to anticipate the early treatment and limit further spread of the disease. Over the last decade, Loop-mediated isothermal amplification (LAMP) assays have become a powerful tool for rapid diagnosis of infectious diseases because of the simplicity of device requirements. Indeed, LAMP is a simple, quick and cost effective Isothermal Nucleic Acid Amplification diagnostic test (INAAT) that has the potential to be used in TB endemic settings of resource-poor countries.\n\nMethods: In the present study, we have developed a simple and rapid TB molecular diagnostic test using a Single-Step Loop-mediated isothermal DNA amplification (SS-LAMP) method for the detection of Mycobacterium tuberculosis complex (MTBC) strains, with a simplified sample preparation procedure, eliminating DNA extraction prior to LAMP amplification, DNA initial denaturation and enzymatic inactivation steps during the amplification process. To perform our in-house SS-LAMP assay, a set of six specific primers was specifically designed to recognize eight distinct regions on the MTBC species-specific repetitive insertion sequence 6110 (IS6110). The amplification of the targeted DNA was carried out under isothermal conditions at 65 \u00b0C within 1 h. Our protocol was firstly optimized using 60 of confirmed MTBC isolates and a recombinant pGEMeasy-IS6110 vector for sensitivity testing. Thereafter, the assay was evaluated on liquefied sputum specimens collected from 157 Moroccan patients suspected of having TB.\n\nResults: Our SS-LAMP developed assay was able to detect MTBC DNA directly from liquefied sputum samples without any prior DNA extraction, denaturation nor the final enzymatic inactivation step. When compared to routinely used L\u00f6wenstein Jensen (LJ) Culture method, our SS-LAMP assay is rapid and showed specificity and sensitivity of 99.14 % and 82.93 % respectively which are within the international standards. In addition, the limit of detection of our assay was found to be as little as 10 copies of bacterial DNA.\n\nConclusion: To our knowledge, this is the first study using a single step LAMP (SS-LAMP) procedure as a rapid, easy to perform and cost effective testing for TB early detection. This innovative assay could be suitable for low-income countries with restricted health equipment facilities.","lamp_id":[{"id":"LPB00624","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"}]},{"id":351,"pmid":19205763,"title":"Development and evaluation of a loop-mediated isothermal amplification method for the rapid detection of Chlamydophila pneumoniae","year":2009,"journal":"European Journal of Clinical Microbiology & Infectious Diseases","authors":"Y Kawai, N Miyashita, F Kishi, M Tabuchi, K Oda, T Yamaguchi, K Kawasaki, T Yamazaki, K Ouchi","doi":"10.1007\/s10096-009-0710-z","country":"Japan","institute":"Kawasaki Medical School","deparment":"Department of Pediatrics","abstract":"We developed a loop-mediated isothermal amplification (LAMP) method to detect Chlamydophila pneumoniae infection. This assay exclusively amplified C. pneumoniae sequences and no cross-reactivity was observed for other Chlamydia species. The detection limit for this assay was found to be ten elementary bodies in 25 min, as observed in a real-time turbidimeter and electrophoretic analysis. The specificity of the LAMP reaction was confirmed by restriction endonuclease analysis, as well as direct sequencing of the amplified product. Among nasopharyngeal swab specimens from 120 patients with acute respiratory tract infections and 40 healthy individuals, the LAMP results showed 100% agreement with the results of real-time polymerase chain reaction (PCR) assays.","lamp_id":[{"id":"LPB00625","pathogen":"Chlamydia pneumoniae CWL029","target":"ompA"}]},{"id":352,"pmid":26664746,"title":"Evaluation of Loop-Mediated Isothermal Amplification Assay for the Detection of Pneumocystis jirovecii in Immunocompromised Patients","year":2015,"journal":"Molecular Biology International","authors":"Preeti Singh, Sundeep Singh, Bijay Ranjan Mirdha, Randeep Guleria, Sanjay Kumar Agarwal, Anant Mohan","doi":"10.1155\/2015\/819091","country":"India","institute":"All India Institute of Medical Sciences","deparment":"Department of Microbiology","abstract":"Pneumocystis pneumonia (PCP) is one of the common opportunistic infection among HIV and non-HIV immunocompromised patients. The lack of a rapid and specific diagnostic test necessitates a more reliable laboratory diagnostic test for PCP. In the present study, the loop-mediated isothermal amplification (LAMP) assay was evaluated for the detection of Pneumocystis jirovecii. 185 clinical respiratory samples, including both BALF and IS, were subjected to GMS staining, nested PCR, and LAMP assay. Of 185 respiratory samples, 12\/185 (6.5%), 41\/185 (22.2%), and 49\/185 (26.5%) samples were positive by GMS staining, nested PCR, and LAMP assay, respectively. As compared to nested PCR, additional 8 samples were positive by LAMP assay and found to be statistically significant (p < 0.05) with the detection limit of 1 pg. Thus, the LAMP assay may serve as a better diagnostic tool for the detection of P. jirovecii with high sensitivity and specificity, less turn-around time, operational simplicity, single-step amplification, and immediate visual detection.","lamp_id":[{"id":"LPB00626","pathogen":"Pneumocystis jirovecii","target":"18S rRNA"}]},{"id":353,"pmid":32412826,"title":"Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Rapid and Specific Identification of Carbapenem-Resistant Acinetobacter baumannii Strains Harboring blaOXA-23, and the Epidemiological Survey of Clinical Isolates","year":2020,"journal":"Microbial Drug Resistance","authors":"Puyuan Li, Wenkai Niu, Yun Fang, Dayang Zou, Huiying Liu, Yanhong Qin, Jing Zheng, Xiuyun Yin, Fengjiang Li, Yannan Liu, Xin Yuan, Liuyu Huang, Changqing Bai","doi":"10.1089\/mdr.2019.0441","country":"","institute":"","deparment":"","abstract":"Acinetobacter baumannii is an important nosocomial pathogen in hospital-acquired infections, and carbapenem resistance has been increasingly observed worldwide. Oxacillinase production by blaOXA-23 is a predominant and prevalent carbapenem resistance mechanism of A. baumannii, especially in China. Rapid and specific detection of blaOXA-23 may offer valuable insight for administration of directed antimicrobial therapy. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP)-based method for identifying carbapenem-resistant A. baumannii (CRAB) harboring the blaOXA-23 gene. High-specificity primers for screening blaOXA-23 were designed and synthesized, and the LAMP reactions were performed. Clinical A. baumannii strains isolated from the Former 307th Hospital of People's Liberation Army were used to determine the sensitivity and specificity of this method compared with those of phenotypic antimicrobial susceptibility testing and the traditional PCR method. Multilocus sequence typing (MLST) was performed to investigate the epidemiology of the A. baumannii bacterial population. Compared with antimicrobial susceptibility testing, the sensitivity and specificity of LAMP in detecting blaOXA-23 were 88.4% and 97.7%, respectively. However, the LAMP method is much simpler and less time-consuming (within 60 minutes) than conventional PCR and phenotypic susceptibility testing. The 113 isolates could be clustered into 30 sequence types, and most strains (83\/113) belonged to clonal complex (CC) 92, which is also the dominant CC in China. The LAMP-based method detected blaOXA-23 in a simpler manner and could provide rapid results for identifying CRAB. Consequently, blaOXA-23 may serve as a surrogate marker for the presence of CRAB in patients with serious infections in clinical practice.","lamp_id":[{"id":"LPB00627","pathogen":"Acinetobacter baumannii","target":"blaOXA-23"}]},{"id":354,"pmid":35211469,"title":"Loop-Mediated Isothermal Amplification Coupled With Nanoparticle-Based Lateral Biosensor for Rapid, Sensitive, and Specific Detection of Bordetella pertussis","year":2022,"journal":"Frontiers in Bioengineering and Biotechnology","authors":"Chunrong Sun, Fei Xiao, Jin Fu, Xiaolan Huang, Nan Jia, Zheng Xu, Yi Wang, Xiaodai Cui","doi":"10.3389\/fbioe.2021.797957","country":"","institute":"","deparment":"","abstract":"Bordetella pertussis is the most frequent causative agent for pertussis, which is a highly contagious disease. Here, we developed a method based on loop-mediated isothermal amplification (LAMP) and nanoparticle-based lateral flow biosensor (LFB) for the timely diagnosis of B. pertussis infections. A set of six primers was designed for LAMP reactions, and the LAMP results were rapidly and visually indicated using LFB. The recommended condition for the B. pertussis LAMP reactions is 40 min at 66\u00b0C. Our results confirmed that the LAMP-LFB assay could specifically detect B. pertussis and did not cross-react with non-B. pertussis isolates. The sensitivity of the B. pertussis LAMP-LFB assay was 50 fg per reaction. In particular, 108 nasopharyngeal swab (NPS) samples were collected to evaluate the B. pertussis LAMP-LFB assay, and the results were compared with those of the quantitative PCR (qPCR) method. The positive rates of B. pertussis LAMP-LFB and qPCR were 40.7% and 38.8%, respectively, and the agreement between the LAMP-LFB and qPCR results was 98%, with a kappa value of 0.96. The whole process of LAMP-LFB can be completed within 1 h, which is much shorter than that of qPCR, including about 15 min of rapid DNA extraction, 40 min of LAMP reaction, and within 2 min of the LFB test. Collectively, the B. pertussis LAMP-LFB assay developed in this report offers a new option for the rapid, reliable, and simple diagnosis of B. pertussis infections.","lamp_id":[{"id":"LPB00628","pathogen":"Bordetella pertussis Tohama I","target":"PT promoter region"}]},{"id":355,"pmid":27836764,"title":"Direct detection of various pathogens by loop-mediated isothermal amplification assays on bacterial culture and bacterial colony","year":2017,"journal":"Microbial Pathogenesis","authors":"Muxia Yan, Weidong Li, Zhenwen Zhou, Hongxia Peng, Ziyan Lu, Ling Xu","doi":"10.1016\/j.micpath.2016.10.025","country":"China","institute":"Guangzhou Women and Children's Medical Centre","deparment":"Department of Haematology","abstract":"In this work, loop-mediated isothermal amplification based detection assay using bacterial culture and bacterial colony for various common pathogens direct detection had been established, evaluated and further applied. A total of five species of common pathogens and nine detection targets (tlh, tdh and trh for V. Parahaemolyticus, rfbE, stx1 and stx2 for E. coli, oprI for P. aeruginosa, invA for Salmonella and hylA for L. monocytogenes) were performed on bacterial culture and bacterial colony LAMP. To evaluate and optimize this assay, a total of 116 standard strains were included. Then, for each detected targets, 20 random selected strains were applied. Results were determined through both visual observation of the changed color by naked eye and electrophoresis, which increased the accuracy of survey. The minimum adding quantity of each primer had been confirmed, and the optimal amplification was obtained under 65 \u00b0C for 45 min with 25 \u03bcl reaction volume. The detection limit of bacterial culture LAMP and PCR assay were determined to be 10^2 and 10^4 or 10^5 CFU\/reaction, respectively. No false positive amplification was observed when subjecting the bacterial -LAMP assay to 116 reference strains. This was the first report of colony-LAMP and culture-LAMP assay, which had been demonstrated to be a fast, reliable, cost-effective and simple method on detection of various common pathogens.","lamp_id":[{"id":"LPB00629","pathogen":"Pseudomonas aeruginosa","target":"oprI"}]},{"id":356,"pmid":16672435,"title":"Development and evaluation of a loop-mediated isothermal amplification method for rapid diagnosis of Bordetella pertussis infection","year":2006,"journal":"Journal of Clinical Microbiology","authors":"Kazunari Kamachi, Hiromi Toyoizumi-Ajisaka, Kohei Toda, Sann Chan Soeung, Svay Sarath, Ya Nareth, Yoshinobu Horiuchi, Kazunobu Kojima, Motohide Takahashi, Yoshichika Arakawa","doi":"10.1128\/JCM.44.5.1899-1902.2006","country":"Japan","institute":"National Institute of Infectious Diseases,","deparment":"Department of Bacterial Pathogenesis and Infection Control","abstract":"We developed a loop-mediated isothermal amplification (LAMP) method to detect Bordetella pertussis infection. This LAMP assay detected B. pertussis with high sensitivity, but not other Bordetella species. Among nasopharyngeal swab samples from subjects with suspected pertussis, LAMP results showed a high level of agreement with results of conventional PCR. This method is a rapid, sensitive, and specific method for diagnosis of B. pertussis infection even in clinical laboratories with no specific equipment.","lamp_id":[{"id":"LPB00630","pathogen":"Bordetella pertussis Tohama I","target":"PT promoter region"}]},{"id":357,"pmid":17314429,"title":"Detection of Haemophilus influenzae by loop-mediated isothermal amplification (LAMP) of the outer membrane protein P6 gene","year":2007,"journal":"Japanese Journal of Infectious Diseases","authors":"Hirotaka Torigoe, Mitsuko Seki, Yoshihisa Yamashita, Atsuto Sugaya, Masao Maeno","doi":"","country":"Japan","institute":"Nihon University School of Dentistry","deparment":"Department of Oral Health Sciences","abstract":"It is difficult and time-consuming to distinguish Haemophilus influenzae from the genotypically similar Haemophilus parainfluenzae, which is a commensal of the human oral cavity. The novel nucleic acid amplification technique of loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63 degrees C) with high specificity, efficiency, and rapidity, was evaluated for H. influenzae detection. A H. influenzae-specific LAMP primer set was designed for the outer membrane protein P6 gene. Primer set specificity was validated using 4 Haemophilus spp. and 13 other species. Within 60 min, LAMP detected 100 or more copies of purified DNA with a sensitivity that was 10-fold higher than that of conventional PCR. This method can be used to differentiate H. influenzae from H. parainfluenzae strains. Thus, LAMP may represent a sensitive and reliable means of diagnosing H. influenzae infection.","lamp_id":[{"id":"LPB00631","pathogen":"Haemophilus influenzae Rd KW20","target":"OMP P6 (pal)"}]},{"id":358,"pmid":35513820,"title":"Detection of Haemophilus influenzae by loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor assay","year":2022,"journal":"BMC Microbiology","authors":"Qilong Cao, Shaoshuai Liang, Feng Lin, Jun Cao, Lin Wang, Hui Li, Mengyang Liu, Yajuan Wang, Lijun Zhao, Xiaolong Cao, Yan Guo","doi":"10.1186\/s12866-022-02547-5","country":"China","institute":"Key Laboratory of Biomedical Information Engineering of Ministry of Education","deparment":"Biomedical Informatics & Genomics Center","abstract":"Background: Haemophilus influenzae was the most aggressive pathogen and formed a major cause of bacterial meningitis and pneumonia in young children and infants, which need medical emergency requiring immediate diagnosis and treatment. However, From isolation to identification of H. influenzae, the traditional diagnose strategy was time-consuming and expensive. Therefore, the establishment of a convenient, highly sensitive, and stable detection system is urgent and critical.\n\nResults: In this study, we used a combined method to detect H. influenzae. Six specific primers were designed on the basis of outer membrane protein P6 gene sequence of H. influenzae. The reaction condition such as the optimum temperature was 65\u2103, and the optimum reaction time was 30 min, respectively. Through the loop-mediated isothermal amplification (LAMP) in combination with nanoparticle-based lateral flow biosensor (LFB), the sensitivity of LAMP-LFB showed 100 fg was the lowest genomic DNA templates concentration in the pure cultures. Meanwhile, the specificity of H. influenzae-LAMP-LFB assay showed the exclusive positive results, which were detected in H. influenzae templates. In 55 clinical sputum samples, 22 samples were positive with LAMP-LFB method, which was in accordance with the traditional culture and Polymerase Chain Reaction (PCR) method. The accuracy in diagnosing H. influenzae with LAMP-LFB could reach 100%, compared to culture and PCR method, indicating the LAMP-LFB had more advantages in target pathogen detection.\n\nConclusions: Taken together, LAMP-LFB could be used as an effective diagnostic approach for H. influenzae in the conditions of basic and clinical labs, which would allow clinicians to make better informed decisions regarding patient treatment without delay.","lamp_id":[{"id":"LPB00632","pathogen":"Haemophilus influenzae Rd KW20","target":"OMP P6 (pal)"}]},{"id":359,"pmid":35873146,"title":"Loop-Mediated Isothermal Amplification Coupled With Nanoparticle-Based Biosensor: A Rapid and Sensitive Method to Detect Mycoplasma pneumoniae","year":2022,"journal":"Frontiers in Cellular and Infection Microbiology","authors":"Fei Xiao, Juan Zhou, Chunrong Sun, Xiaolan Huang, Baoying Zheng, Jin Fu, Nan Jia, Zheng Xu, Xiaodai Cui, Yi Wang","doi":"10.3389\/fcimb.2022.882855","country":"China","institute":"Capital Institute of pediatrics","deparment":"Experimental research center","abstract":"Mycoplasma pneumoniae (MP), the causative agent of MP pneumonia (MPP), has posed a substantial burden to public health owing to a lack of rapid and effective diagnostic methods. Here, we designed a loop-mediated isothermal amplification (LAMP)-based assay, termed LAMP, combined with a nanoparticle-based lateral flow biosensor (LAMP-LFB) for rapid and sensitive diagnosis of MP.-LAMP-LFB included a set of six primers targeting the community-acquired respiratory distress syndrome (CARDS) toxin gene and was performed optimally at 63\u00b0C for only 30 min. The resulting LAMP products could be visually indicated by LFB within 2 min, thus the whole process could be accomplished within an hour. MP-LAMP-LFB's sensitivity was 50 fg per reaction, which was in complete accordance with these results obtained from real-time turbidity and visual detection reagent (VDR). MP-LAMP-LFB had no cross-reactivity with other pathogens that had similar clinical presentations. Our assay was further validated using 100 nasopharyngeal swab samples collected from children suspected of MPP, and the result was compared with the real-time PCR method. With a positive rate of 50%, the data indicated that MP-LAMP-LFB is a sensitive test for MP detection in clinical settings. Collectively, the MP-LAMP-LFB assay targeting the CARDS toxin gene was a rapid, highly sensitive, and specific test that could be widely applied in point-of-care settings and basic medical facilities in rural areas.","lamp_id":[{"id":"LPB00633","pathogen":"Mycoplasma pneumoniae","target":"CARDS toxin"}]},{"id":360,"pmid":22370920,"title":"Detection of Mycoplasma pneumoniae by loop-mediated isothermal amplification (LAMP) assay and serology in pediatric community-acquired pneumonia","year":2012,"journal":"Journal of Infection and Chemotherapy","authors":"Kensei Gotoh, Naoko Nishimura, Yasunori Ohshima, Yasuko Arakawa, Haruki Hosono, Yasuto Yamamoto, Yasushi Iwata, Kazumasa Nakane, Keiji Funahashi, Takao Ozaki","doi":"10.1007\/s10156-012-0388-5","country":"Japan","institute":"Konan Kosei Hospital","deparment":"Department of Pediatrics","abstract":"Rapid diagnosis of Mycoplasma pneumoniae pneumonia is required for treatment with effective antimicrobial agents without delay; however, this capacity has not yet been established in clinical practice. Recently, a novel nucleic acid amplification method termed loop-mediated isothermal amplification (LAMP) has been used to rapidly diagnose various infectious diseases. In this study, we prospectively evaluated the efficacy of the LAMP assay to rapidly diagnose M. pneumoniae pneumonia in clinical practice. Three hundred sixty-eight children (median age, 3.8 years; range, 0.1-14.3 years) admitted to our hospital between April 2009 and March 2010 for community-acquired pneumonia were enrolled in this study. We obtained throat swabs on admission to detect M. pneumoniae DNA and paired serum samples on admission and at discharge to assay M. pneumoniae antibody titers. M. pneumoniae pneumonia was diagnosed by either a positive LAMP assay or a fourfold or greater increase in antibody titer. Overall, 46 children (12.5% of the patients with pneumonia) were diagnosed with M. pneumoniae pneumonia; of these, 27 (58.7%) were aged less than 6 years. Of the aforementioned 46 children, 38 (82.6%) and 37 (80.4%) were identified by LAMP and serology, respectively. When the results of serology were taken as the standard, the sensitivity and specificity and positive and negative predictive values of the LAMP assay were 78.4%, 97.3%, 76.3%, and 97.6%, respectively. We concluded the LAMP assay may be useful for rapid diagnosis of M. pneumoniae pneumonia.","lamp_id":[{"id":"LPB00634","pathogen":"Mycoplasma pneumoniae","target":"SDC1 repetitive sequence"}]},{"id":361,"pmid":25798436,"title":"Survey and Rapid Detection of Bordetella pertussis in Clinical Samples Targeting the BP485 in China","year":2015,"journal":"Frontiers in Public Health","authors":"Wei Liu, Yinghua Xu, Derong Dong, Huan Li, Xiangna Zhao, Lili Li, Ying Zhang, Xiao Wei, Xuesong Wang, Simo Huang, Ming Zeng, Liuyu Huang, Shumin Zhang, Jing Yuan","doi":"10.3389\/fpubh.2015.00039","country":"China","institute":"Academy of Military Medical Sciences","deparment":"Institute of Disease Control and Prevention","abstract":"Bordetella pertussis is an important human respiratory pathogen. Here, we describe a loop-mediated isothermal amplification (LAMP) method for the rapid detection of B. pertussis in clinical samples based on a visual test. The LAMP assay detected the BP485 target sequence within 60 min with a detection limit of 1.3 pg\/\u03bcl, a 10-fold increase in sensitivity compared with conventional PCR. All 31 non-pertussis respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for B. pertussis. To evaluate the application of the LAMP assay to clinical diagnosis, of 105 sputum and nasopharyngeal samples collected from the patients with suspected respiratory infections in China, a total of 12 B. pertussis isolates were identified from 33 positive samples detected by LAMP-based surveillance targeting BP485. Strikingly, a 4.5 months old baby and her mother were found to be infected with B. pertussis at the same time. All isolates belonged to different B. pertussis multilocus sequence typing groups with different alleles of the virulence-related genes including four alleles of ptxA, six of prn, four of tcfA, two of fim2, and three of fim3. The diversity of B. pertussis carrying toxin genes in clinical strains indicates a rapid and continuing evolution of B. pertussis. This combined with its high prevalence will make it difficult to control. In conclusion, we have developed a visual detection LAMP assay, which could be a useful tool for rapid B. pertussis detection, especially in situations where resources are poor and in point-of-care tests.","lamp_id":[{"id":"LPB00635","pathogen":"Bordetella pertussis CS","target":"BP485"}]},{"id":362,"pmid":23852783,"title":"Evaluation of in-house loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of M. tuberculosis in pulmonary specimens","year":2013,"journal":"Journal of Clinical Laboratory Analysis","authors":"Sunil Sethi, Sumeet Singh, Sunil Kumar Dhatwalia, Rakesh Yadav, Abhishek Mewara, Malkit Singh, Rupinder Tewari, Meera Sharma","doi":"10.1002\/jcla.21596","country":"India","institute":"Postgraduate Institute of Medical Education and Research","deparment":"Department of Medical Microbiology","abstract":"Background: Loop-mediated isothermal amplification (LAMP) assay has come forward as a rapid, cost-effective molecular technique for diagnosis of tuberculosis (TB) in developing countries. This study evaluated Mycobacterium tuberculosis-specific in-house LAMP assay targeting 16s rRNA and compared it with other conventional tests and nucleic acid amplification assay (IS6110 PCR).\n\nMethods: A total of 133 sputum specimens (103 from suspected pulmonary TB cases and 30 from non-TB controls) were subjected to conventional tests, IS6110 PCR and 16s rRNA LAMP assay.\n\nResults: Of the 103 patients, the maximum number of cases were found to be positive by LAMP assay, that is, in 87 (84.5%) patients, followed by culture positive in 78 (75.7%), IS6110 PCR in 74 (71.8%), and smear positive in 70 (67.9%) patients. Of the 83 smear positive and\/or culture positive cases, LAMP detected 77 (92.77%) cases, and was found to be superior to IS6110 PCR, which could detect 69 (83.1%) cases; a concordance of 0.6 was obtained between the two tests using kappa statistics.\n\nConclusion: Overall, LAMP was simple and efficacious for early diagnosis of smear positive, culture positive cases as well as for confirmation of smear negative, culture negative cases, and was found to be superior to IS6110 PCR.","lamp_id":[{"id":"LPB00636","pathogen":"Mycobacterium tuberculosis","target":"16S rRNA"}]},{"id":363,"pmid":26791368,"title":"Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for Rapid Detection of Aspergillus fumigatus","year":2016,"journal":"Journal of Clinical Microbiology","authors":"Qing Tang, Shuguang Tian, Nong Yu, Xi Zhang, Xiaodong Jia, Hongyan Zhai, Qun Sun, Li Han","doi":"10.1128\/JCM.01751-15","country":"P.R China","institute":"Sichuan University","deparment":"Key Laboratory of Bio-resources and Eco-environment of the Ministry of Education","abstract":"Aspergillus fumigatusis a conditional pathogen and the major cause of life-threatening invasive aspergillosis (IA) in immunocompromised patients. The early and rapid detection ofA. fumigatusinfection is still a major challenge. In this study, the new member of the fungal annexin family, annexin C4, was chosen as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific, and sensitive detection ofA. fumigatus The evaluation of the specificity of the LAMP assay that was developed showed that no false-positive results were observed for the 22 non-A. fumigatusstrains, including 5 species of theAspergillusgenus. Its detection limit was approximately 10 copies per reaction in reference plasmids, with higher sensitivity than that of real-time quantitative PCR (qPCR) at 10(2)copies for the same target. Clinical samples from a total of 69 patients with probable IA (n =14) and possible IA (n= 55) were subjected to the LAMP assay, and positive results were found for the 14 patients with probable IA (100%) and 34 patients with possible IA (61.82%). When detection using the LAMP assay was compared with that using qPCR in the 69 clinical samples, the LAMP assay demonstrated a sensitivity of 89.19% and the concordance rate for the two methods was 72.46%. Accordingly, we report that a valuable LAMP assay for the rapid, specific, and simple detection ofA. fumigatusin clinical testing has been developed.","lamp_id":[{"id":"LPB00637","pathogen":"Aspergillus fumigatus","target":"anxC4"}]},{"id":364,"pmid":27045360,"title":"Development of a loop-mediated isothermal amplification method for rapid detection of streptococcal pyrogenic exotoxin B","year":2016,"journal":"Toxicon","authors":"Cuiming Cao, Fang Zhang, Mingyu Ji, Fengyan Pei, Xiujie Fan, Hong Shen, Qingxi Wang, Weihua Yang, Yunshan Wang","doi":"10.1016\/j.toxicon.2016.03.019","country":"China","institute":"Jinan Central Hospital Affiliated to Shandong Universit","deparment":"Medical Research & Laboratory Diagnostic Center","abstract":"We developed a visual loop-mediated isothermal amplification (LAMP) technique to detect the streptococcal pyrogenic exotoxin B (speB) gene. Fifteen strains (from American Type Culture Collection or clinical isolates) were used to determine the specificity and sensitivity of the LAMP assay. Clinical samples were collected from 132 patients with suspected Streptococcus pyogenes (S. pyogenes) infection to verify the feasibility of the LAMP assay for detection of the speB gene. By using a set of five primers (a pair of outer primers, a pair of inner primers and one loop primer) targeting the speB gene, the amplification reaction was rapidly performed in a regular water bath under isothermal conditions at 63 \u00b0C for approximately 60 min. Only the two S. pyogenes strains showed positive results which were easily observed with the naked eye, and the other strains showed negative results. The detection limit of the LAMP assay was 0.01 ng\/\u03bcl of template, showing higher sensitivity than conventional PCR (with a detection limit of 1.0 ng\/\u03bcl). The detection rate of the speB gene in clinical samples was 71.21% and was consistent with the PCR results. The rapid detection of the speB gene by the LAMP assay is highly specific and sensitive, is simple to perform and cost-effective, and is expected to be a new reliable method for the rapid diagnosis of S. pyogenes infection, that is particularly suitable for rural or community hospitals in developing countries.","lamp_id":[{"id":"LPB00638","pathogen":"Streptococcus pyogenes","target":"speB"}]},{"id":365,"pmid":12791888,"title":"Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples","year":2003,"journal":"Journal of Clinical Microbiology","authors":"Tomotada Iwamoto, Toshiaki Sonobe, Kozaburo Hayashi","doi":"10.1128\/JCM.41.6.2616-2622.2003","country":"Japan","institute":"Kobe Institute of Health","deparment":"Department of Parasitic Agents","abstract":"Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. We used LAMP for detection of Mycobacterium tuberculosis complex, Mycobacterium avium, and Mycobacterium intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) or on a solid medium (Ogawa's medium). Species-specific primers were designed by targeting the gyrB gene, and their specificities were validated on 24 mycobacterial species and 7 nonmycobacterial species. The whole procedure is quite simple, starting with the mixing of all reagents in a single tube, followed by an isothermal reaction during which the reaction mixture is held at 63 degrees C. The resulting amplicons are visualized by adding SYBR Green I to the reaction tube. The only equipment needed for the amplification reaction is a regular laboratory water bath or heat block that furnishes a constant temperature of 63 degrees C. The assay had a detection limit of 5 to 50 copies of purified DNA with a 60-min incubation time. The reaction time could be shortened to 35 min for the species identification of M. tuberculosis complex, M. avium, and M. intracellulare from a solid-medium culture. Residual DNA lysates prepared for the Amplicor assay (Roche Diagnostics GmbH) from 66 sputum specimens were tested in the LAMP assay. Although the sample size used for the latter assay was small, 2.75 micro l of the DNA lysates, it showed a performance comparable with that of the Amplicor assay, which required 50 micro l of the lysates. This LAMP-based assay is simple, rapid, and sensitive; a result is available in 35 min for a solid-medium culture and in 60 min for a liquid-medium culture or for a sputum specimen that contains a corresponding amount of DNA available for testing.","lamp_id":[{"id":"LPB00639","pathogen":"Mycobacterium tuberculosis","target":"gyrB"},{"id":"LPB00640","pathogen":"Mycobacterium avium","target":"gyrB"},{"id":"LPB00641","pathogen":"Mycobacterium intracellulare","target":"gyrB"},{"id":"LPB00642","pathogen":"Mycobacterium","target":"16S rDNA"}]},{"id":366,"pmid":29923972,"title":"Rapid detection of Mycoplasma pneumoniae by loop-mediated isothermal amplification assay","year":2018,"journal":"Medicine","authors":"Xin Yuan, Changqing Bai, Qian Cui, Han Zhang, Jing Yuan, Kaiwen Niu, Yuzhong Feng, Xin Jin, Puyuan Li, Huiying Liu","doi":"10.1097\/MD.0000000000010806","country":"China","institute":"Academy of Military Medical Sciences","deparment":"Department of Respiratory and Critical Care Medicine","abstract":"To improve the diagnosis and treatment of Mycoplasma pneumoniae (Mp) infection and reduce the misuse of antibiotics, we sought to establish a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Mp.Six primers specific for the Mp P1 gene were designed, and the LAMP method was used to rapidly detect Mp. The sensitivity of the LAMP method was determined by serial dilution of the standard Mp strain FH (standard strains of Mycoplasma pneumoniae). Specificity was assessed with 17 common pathogenic microorganisms in the respiratory tract. Patient samples were collected from the Department of Respiratory and Critical Care Medicine at the 307th Hospital of Chinese People's Liberation Army from March 2016 to May 2017, examined prospectively, and compared with diagnosis by quantitative real-time polymerase chain reaction (qRT-PCR).The LAMP assay for Mp detection can be completed within 60 minutes. The minimum detection limit was 39 pg\/\u03bcL, and no cross-reaction was observed with 17 common respiratory tract pathogens. Of the 125 clinical specimens tested, 43 cases were positive by LAMP assay, and 40 cases were positive by qRT-PCR (P = .162). All 43 samples determined as positive by LAMP test were confirmed to be Mp by Mp P1 protein sequencing.The LAMP assay is suitable for rapid detection of Mp. It has high sensitivity and specificity, and the detection results are not inferior to those of qRT-PCR.","lamp_id":[{"id":"LPB00643","pathogen":"Mycoplasma pneumoniae","target":"P1"}]},{"id":367,"pmid":29137710,"title":"Loop-mediated isothermal amplification using self-avoiding molecular recognition systems and antarctic thermal sensitive uracil-DNA-glycosylase for detection of nucleic acid with prevention of carryover contamination","year":2017,"journal":"Analytica Chimica Acta","authors":"Yi Wang, Dongxin Liu, Jianping Deng, Yan Wang, Jianguo Xu, Changyun Ye","doi":"10.1016\/j.aca.2017.10.022","country":"P. R. China","institute":"National Institute for Communicable Disease Control and Prevention","deparment":"State Key Laboratory of Infectious Disease Prevention and Control","abstract":"Loop-mediated isothermal amplification (LAMP) is the most popular technique to amplify nucleic acid sequence without the use of temperature cycling. However, LAMP is often confounded by false-positive results, arising from interactions between (hetero-dimer) or within (self-dimerization) primers, off-target hybrids and carryover contaminants. Here, we devised a new LAMP technique that is self-avoiding molecular recognition system (SAMRS) components and antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) enzyme-assisted, termed AUDG-SAMRS-LAMP. Incorporating SAMRS components into 3'-ends of LAMP primers can improve assay's specificity, which completely prevents the non-specific amplification yielding from off-target hybrids and undesired interactions between or within primers. Adding AUDG into reaction mixtures can effectively eliminate the false-positive results arising from carryover contamination, thus the genuine positive reactions are generated from the amplification of target templates. Furthermore, AUDG-SAMRS-LAMP results are confirmed using a new analysis strategy, which is developed for detecting LAMP amplicons by lateral flow biosensor (LFB). Only a single labeled primer is required in the analysis system, thus the false positive results arising from hybridization (the labeled primer and probe, or between two labeled primers) are avoided. Hence, the SAMRS components, AUDG and LFB convert traditional LAMP from a technique suited for the research laboratory into one that has practical value in the field of diagnosis. Human Tuberculosis (TB) is caused by infection with members of Mycobacterium tuberculosis complex (MTC), which are detected by the AUDG-SAMRS-LAMP technique to demonstrate the availability of target analysis. The proof-of-concept method can be reconfigured to detect various nucleic acids by redesigning the specific primers.","lamp_id":[{"id":"LPB00644","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"}]},{"id":368,"pmid":28940325,"title":"Evaluation of the RT\u2010LAMP and LAMP methods for detection of Mycobacterium tuberculosis","year":2017,"journal":"Journal of Clinical Laboratory Analysis","authors":"Dandan Wu, Jiwen Kang, Baosheng Li, Dianxing Sun","doi":"10.1002\/jcla.22326","country":"China","institute":"Chengde Medical University","deparment":"","abstract":"Background: The current methods for detecting Mycobacterium tuberculosis (Mtb) are not clinically optimal. Standard culture methods (SCMs) are slow, costly, or unreliable, and loop-mediated isothermal amplification (LAMP) cannot differentiate live Mtb.\n\nMethods: This study compared reverse transcription (RT)-LAMP, LAMP, and an SCM for detecting Mtb. A first experiment tested the sensitivity and specificity of primers for 9 species of Mycobacterium (H37Rv, M. intracellulare, M. marinum, M. kansasii, M. avium, M. flavescens, M. smegmatis, M. fortuitum, and M. chelonae); and 3 non-Mycobacterium species (Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae). A second experiment tested sputum specimens for the presence of Mtb, from 100 patients with tuberculosis (clinical) and 22 from patients without tuberculosis (control), using Roche solid culture (SCM), LAMP, and RT-LAMP. In the clinical samples.\n\nResults: The rates of positivity for Mtb of the SCM, LAMP, and RT-LAMP methods were 88%, 92%, and 100%, respectively. The difference in detection rate was significant between RT-LAMP and SCM, but RT-LAMP and LAMP were comparable. In the control group, the detection rates were nil for all three methods.\n\nConclusion: The specificities of the methods were similar. The sensitivity of RT-LAMP was ~10-fold higher than that of LAMP for detecting Mtb. Unlike LAMP, RT-LAMP could identify viable bacteria, and was able to detect a single copy of Mtb. Among SCM, LAMP, and RT-LAMP, the latter is the most suitable for wide use in the lower-level hospitals and clinics of China for detecting Mtb in sputum samples.","lamp_id":[{"id":"LPB00645","pathogen":"Mycobacterium tuberculosis H37Rv","target":"16S rRNA"}]},{"id":369,"pmid":33046776,"title":"Ultrasensitive detection of Mycobacterium tuberculosis by a rapid and specific probe-triggered one-step, simultaneous DNA hybridization and isothermal amplification combined with a lateral flow dipstick","year":2020,"journal":"Scientific Reports","authors":"Wansadaj Jaroenram, Jantana Kampeera, Narong Arunrut, Sarawut Sirithammajak, Sarinya Jaitrong, Kobporn Boonnak, Pakapreud Khumwan, Therdsak Prammananan, Angkana Chaiprasert, Wansika Kiatpathomchai","doi":"10.1038\/s41598-020-73981-6","country":"Thailand","institute":"National Center for Genetic Engineering and Biotechnology (BIOTEC)","deparment":"Bioengineering and Sensing Technology Research Team","abstract":"Mycobacterium tuberculosis (Mtb) is an insidious scourge that has afflicted millions of people worldwide. Although there are many rapid methods to detect it based on loop-mediated isothermal amplification (LAMP) and a lateral flow dipstick (LFD), this study made further improvements using a new set of primers to enhance LAMP performance and a novel DNA probe system to simplify detection and increase specificity. The new probe system eliminates the post-LAMP hybridization step typically required for LFD assays by allowing co-hybridization and amplification of target DNA in one reaction while preventing self-polymerization that could lead to false-positive results. The improved assay was named Probe-Triggered, One-Step, Simultaneous DNA Hybridization and LAMP Integrated with LFD (SH-LAMP-LFD). SH-LAMP-LFD was simpler to perform and more sensitive than previously reported LAMP-LFD and PCR methods by 100 and 1000 times, respectively. It could detect a single cell of Mtb. The absence of cross-reactivity with 23 non-TB bacteria, and accurate test results with all 104 blind clinical samples have highlighted its accuracy. Its robustness and portability make SH-LAMP-LFD suitable for users in both low and high resource settings.","lamp_id":[{"id":"LPB00646","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"}]},{"id":370,"pmid":32950563,"title":"Rapid detection of rifampicin-resistant Mycobacterium tuberculosis, based on isothermal DNA amplification and DNA chromatography","year":2020,"journal":"Journal of Microbiological Methods","authors":"Yutaka Takarada, Takuya Kodera, Kumi Kobayashi, Chie Nakajima, Mitsuo Kawase, Yasuhiko Suzuki","doi":"10.1016\/j.mimet.2020.106062","country":"Japan","institute":"TBA Co","deparment":"","abstract":"Rapid and easy detection of nucleotide point mutations in bacterial pathogens associated with drug resistance is essential for the proper use of antimicrobials. Here, we developed a rapid and simple method for the detection of mutations using Loop-mediated isothermal amplification (LAMP) combined with the single-tag hybridization (STH) chromatographic printed array strips (PAS) method. This procedure is able to detect four mutations (C1349 T, A1295C, G1303 T, A1304 T) in Rifampicin Resistance Determining Region (RRDR) of rifampicin-resistant Mycobacterium tuberculosis (RR-TB), simultaneously. LAMP reactions contained a LAMP primer and eight allele-specific primers for each mutation. The allele-specific primers products were detected by nucleic acid chromatography using PAS. Four detection lines were detected there, one of which was detected at different positions depend on the wild type and the mutant type. We carried out the four mutations detection using 31 genomic DNA (2 A1295T, 1 G1303 T, 6 A1304 T, 22 C1349 T) from clinical isolate. The mutations have been confirmed by sequence analysis. The detection results were completely consistent with the sequence analysis. In the present study, four mutations could be detected, but only 60% of RR-TB could be detected with these four. It is expected that the detection rate will increase by adding more mutant primers. The combined LAMP and STH chromatographic PAS method is a simple and rapid method for detecting point mutations in clinical isolates as a point-of-care testing (POCT) technique. In addition, it does not require special equipment and can meet the demand in areas where drug-resistant bacteria are endemic, such as developing countries.","lamp_id":[{"id":"LPB00647","pathogen":"Mycobacterium tuberculosis H37Rv","target":"rpoB"}]},{"id":371,"pmid":32197601,"title":"Pneumocystis jirovecii colonization and its association with pulmonary diseases: a multicenter study based on a modified loop-mediated isothermal amplification assay","year":2020,"journal":"BMC Pulmonary Medicine","authors":"Ting Xue, Zhuang Ma, Fan Liu, Weiqin Du, Li He, Jinyan Wang, Chunli An","doi":"10.1186\/s12890-020-1111-4","country":"P.R China","institute":"China Medical University","deparment":"Department of Microbiology and Parasitology","abstract":"Background: Pneumocystis jirovecii (P. jirovecii) is an opportunistic fungal pathogen and the role of its colonization in pulmonary diseases has become a popular focus in recent years. The aim of this study was to develop a modified loop-mediated isothermal amplification (LAMP) assay for detection of Pneumocystis jirovecii (P. jirovecii) DNA amongst non-HIV patients with various pulmonary diseases and use it to examine the prevalence and assess the association of P. jirovecii colonization with clinical characteristics of these diseases.\n\nMethods: We modified the previously reported LAMP assay for P. jirovecii by adding real-time detection. This method was used to detect P. jirovecii colonization in pulmonary samples collected from 403 non-HIV patients with various pulmonary diseases enrolled from 5 hospitals in China. We determined the prevalence of P. jirovecii colonization in 7 types of pulmonary diseases and assessed the association of P. jirovecii colonization with clinical characteristics of these diseases.\n\nResults: The modified LAMP assay showed no cross-reactivity with other common pulmonary microbes and was 1000 times more sensitive than that of conventional PCR. Using the modified LAMP assay, we detected P. jirovecii colonization in 281 (69.7%) of the 403 patients enrolled. P. jirovecii colonization was more common in interstitial lung diseases than in chronic obstructive pulmonary disease (COPD) (84.6% vs 64.5%, P < 0.05). Patients with acute exacerbation of COPD had a higher prevalence of P. jirovecii colonization compared to patients with stabilized COPD (67.4% vs 43.3%, P < 0.05). P. jirovecii colonization was associated with decreased pulmonary function, increased levels of 1,3-\u03b2-D-glucan and C-reactive protein, and decreased levels of CD4+ T-cell counts (P < 0.05 for each). Approximately 70% of P. jirovecii colonized patients had confections with other fungi or bacteria.\n\nConclusions: We developed a modified LAMP assay for detecting P. jirovecii. Our multi-center study of 403 patients supports that P. jirovecii colonization is a risk factor for the development of pulmonary diseases and highlights the need to further study the pathogenesis and transmission of P. jirovecii colonization in pulmonary diseases.","lamp_id":[{"id":"LPB00648","pathogen":"Pneumocystis jirovecii","target":"16S rRNA"}]},{"id":372,"pmid":32655515,"title":"Rapid Detection to Differentiate Hypervirulent Klebsiella pneumoniae (hvKp) From Classical K. pneumoniae by Identifying peg-344 With Loop-Mediated Isothermal Amplication (LAMP)","year":2020,"journal":"Frontiers in Microbiology","authors":"Wenjian Liao, Dan Long, Qisen Huang, Dandan Wei, Xiaobing Liu, Lagen Wan, Yuling Feng, Wei Zhang, Yang Liu","doi":"10.3389\/fmicb.2020.01189","country":"China","institute":"The First Affiliated Hospital of Nanchang University","deparment":"Department of Respiratory Medicine","abstract":"Objectives: To establish a rapid molecular diagnostics of hvKp using the peg-344 loop-mediated isothermal amplification technique (LAMP).\n\nMethods: In all, 28 K. pneumoniae strains isolated from the blood of patients were used for the peg-344 LAMP. K. pneumoniae NTUH-K2044 and K. pneumoniae ATCC700603 were used as positive control and negative control, respectively. For comparison, all the results were detected in a polymerase chain reaction (PCR), which was considered the gold standard for the detection of the gene. Mouse lethality assay, and Serum killing assay were also used to determine the virulence phenotype of K. pneumoniae.\n\nResults: We determined the specificity and sensitivity of the primers for peg-344 detection in the LAMP reactions. This LAMP assay was able to specifically differentiate hvKp from classical K. pneumoniae (cKp) at 65\u00b0C, which was 100-fold more sensitive than a PCR assay for peg-344 detection. The virulence phenotype of K. pneumoniae detected by LAMP was as precise as by Mouse lethality assay and Serum killing assay.\n\nConclusion: The LAMP assay is easy to perform and rapid. Therefore, it can be routinely applied to differentiate hvKp from cKp in the clinical laboratory.","lamp_id":[{"id":"LPB00649","pathogen":"Klebsiella pneumoniae subsp. pneumoniae NTUH-K2044","target":"peg-344"}]},{"id":373,"pmid":32409020,"title":"Loop-mediated isothermal amplification (LAMP) in the respiratory specimens for the diagnosis of pediatric pulmonary tuberculosis: A pilot study","year":2020,"journal":"Journal of Infection and Chemotherapy","authors":"K S Sreedeep, Sunil Sethi, Rakesh Yadav, Pankaj C Vaidya, Suresh Kumar Angurana, Aastha Saini, Nancy Mehra, Meenu Singh","doi":"10.1016\/j.jiac.2020.03.019","country":"China","institute":"Postgraduate Institute of Medical Education and Research (PGIMER)","deparment":"Department of Pediatrics","abstract":"Background: Loop-mediated isothermal amplification (LAMP) assay is a novel molecular diagnostic technique that can be used for the diagnosis of tuberculosis (TB). It is most suited for developing countries as it is rapid, inexpensive, highly sensitive, requiring minimal infrastructure, training and manpower. Studies in pediatric TB are lacking. We evaluated LAMP in the diagnosis of pediatric pulmonary TB.\n\nMethodology: This was a cross-sectional analytical study conducted at a tertiary care teaching hospital in North India from July 2014 to June 2015 involving 60 children with suspected pulmonary TB. Respiratory specimens (sputum, gastric lavage, bronchoalveolar lavage and\/or endotracheal aspirates) were collected and subjected to BACTEC MGIT 960 culture, IS6110 PCR, and LAMP assay targeting IS6110 gene of Mycobacterium tuberculosis.\n\nResults: Thirty seven children had confirmed and probable TB according to the composite reference standard (CRS). Among all the 3 tests used for diagnosis of Pulmonary TB, LAMP had highest sensitivity (37.8%) followed by PCR (27%), and culture (21.6%) when compared against the predefined CRS. Culture had maximum specificity of 100%; and PCR, and LAMP had specificity of 95-96%. The sensitivity, specificity, PPV, and NPV of LAMP against culture as reference standard were 75%, 72.4%, 42.9%, and 91.3% respectively. Similarly sensitivity, specificity, PPV, and NPV of PCR against culture as reference standard were 75%, 86.2%, 60%, and 86.2% respectively. On combining LAMP with culture, sensitivity increased to 45.7% (7.8% increase, p = 0.04).\n\nConclusion: We noted that LAMP had highest sensitivity when compared to culture and PCR and comparable specificity.","lamp_id":[{"id":"LPB00650","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"}]},{"id":374,"pmid":32266166,"title":"Validation of a Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Invasive Pneumococcal Disease","year":2020,"journal":"Frontiers in Cellular and Infection Microbiology","authors":"H\u00e9ctor David de Paz, Pedro Brotons, Cristina Esteva, Carmen Mu\u00f1oz-Almagro","doi":"10.3389\/fcimb.2020.00115","country":"Spain","institute":"Institut de Recerca Pediatrica","deparment":"Department of Molecular Microbiology","abstract":"Current molecular PCR-based techniques used for detecting Streptococcus pneumoniae, the causative pathogen of invasive pneumococcal disease (IPD), are accurate but have a run time of several hours. We aimed to develop and validate a novel real-time loop mediated amplification (LAMP) assay for rapid detection of pneumococcus in normally sterile samples with accuracy comparable to a gold standard real-time PCR. Conserved regions of lytA were used for the design of the LAMP test. Analytical validation included assessment of linearity, limit of detection (LOD), intra-assay and inter-assay precision and analytical specificity, which was evaluated by using reference strain S. pneumoniae R6 and a quality control panel. Clinical performance was assessed on all samples collected from children with suspicion of IPD attended in Hospital Sant Joan de Deu (Barcelona, Spain) during the period April-September 2015. Fresh samples were analyzed after DNA extraction. The following values of analytical parameters were determined: linearity within the range 10^8-10^4 copies\/mL; limit of detection, 5\u00b710^3 copies\/mL; intra- and inter-assay precision measured by mean coefficient of variance, 3.61 and 6.59%; analytical specificity, 9\/9 pathogens similar to S. pneumoniae and 14\/14 strains of different S. pneumoniae serotypes correctly identified as negative and positive results, respectively. Diagnostic sensitivity and specificity values were 100.0 and 99.3%. Median time of DNA amplification was 15 min. The new LAMP assay showed to have similar accuracy as PCR while being 5-fold faster and could become a useful diagnostic tool for early diagnosis of IPD.","lamp_id":[{"id":"LPB00651","pathogen":"Streptococcus pneumoniae","target":"lytA"}]},{"id":375,"pmid":31970983,"title":"Polymerization-Based Amplification for Target-Specific Colorimetric Detection of Amplified Mycobacterium tuberculosis DNA on Cellulose","year":2020,"journal":"ACS Sensors","authors":"Emma H Yee, Hadley D Sikes","doi":"10.1021\/acssensors.9b02424","country":"Singapore","institute":"Singapore-MIT Alliance for Research and Technology (SMART) Centre","deparment":"","abstract":"Loop-mediated isothermal amplification (LAMP) is an appealing method for low-cost, point-of-care nucleic acid diagnostic assays due to high sensitivity, minimal equipment requirements, and compatibility with user-friendly colorimetric detection methods. The enhanced sensitivity LAMP offers comes with vulnerability to cross-contamination, where negative samples are exposed to minute amounts of nucleic acids from positive samples. These amounts are insignificant in less sensitive amplification methods, but visible when LAMP is paired with common colorimetric methods. Here, we examined the use of eosin photopolymerization, a tunable reaction, for colorimetric detection of LAMP products to reduce this false positive risk. Using eosin and biotin end-labeled primers, we successfully amplified target regions of the Mycobacterium tuberculosis (MTB) genome using PCR and LAMP, captured amplicons on streptavidin-coated cellulose, and detected DNA targets via eosin photopolymerization, producing a bright pink color only if MTB DNA was present in the sample. Consistent with previous reports, the LAMP-based method exhibited high background signal, but tuning the illumination time for the photopolymerization reaction allowed readouts from samples with no added MTB DNA to remain blank and visually distinct from pink positives. This method yielded limits of detection of 30 and 300 copies\/\u03bcL for LAMP and PCR amplification, respectively. When confronted with boiled MTB culture samples, this method gave clear positive readouts, compared to negligible signal from other Mycobacterium boiled culture samples. This new method of LAMP colorimetric detection has the potential to increase the utility of LAMP as a nucleic acid assay technique by mitigating sensitivity to cross-contamination.","lamp_id":[{"id":"LPB00652","pathogen":"Mycobacterium tuberculosis H37Rv","target":"gyrB"}]},{"id":376,"pmid":31807946,"title":"Development of loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor assay for Mycoplasma pneumoniae detection","year":2019,"journal":"AMB Express","authors":"Yacui Wang, Yi Wang, Weiwei Jiao, Jieqiong Li, Shuting Quan, Lin Sun, Yonghong Wang, Xue Qi, Xingyun Wang, Adong Shen","doi":"10.1186\/s13568-019-0921-3","country":"China","institute":"National Key Discipline of Pediatrics (Capital Medical University)","deparment":"Key Laboratory of Major Diseases in Children","abstract":"Mycoplasma pneumoniae (MP) is one of the most common pathogens causing respiratory tract infection, especially for community-acquired pneumonia (CAP) in school-age children. There was considerable amount of studies on loop-mediated isothermal amplification (LAMP) assay for MP detection. However, the result interpretation of these developed LAMP assays was sophisticated and subjective. Therefore, we developed and evaluated a LAMP coupled with nanoparticle-based lateral flow biosensor (LFB) assay (LAMP-LFB) for simple, reliable, and objective identification of MP (MP-LAMP-LFB). Six primers specific to P1 gene of MP were designed, and the preferred temperature for this assay was confirmed to be 65 \u00b0C. The amplification products could be visually interpreted by LFB within 2 min. The MP-LAMP-LFB assay specifically identified DNA templates of MP, and no cross-reactivity with other pathogens was obtained. The limit of the detection for this assay was 600 fg of DNA templates in pure cultures, which was in complete accordance with colorimetric indicator detection and agarose gel electrophoresis analysis. This assay was applied to 209 oropharyngeal swab specimens collected from children with acute respiratory tract infection for clinical evaluation, and compared to real-time PCR detection. Using the LAMP-LFB and real-time PCR assay, the positive rates of MP were 47.8% and 31.6%, respectively. Results suggested that the LAMP-LFB assay displayed high sensitivity compared to real-time PCR method. In summary, LAMP-LFB assay established here was a simple, objective, and sensitive assay for MP detection, which can be widely applied in clinical settings, especially in rural areas.","lamp_id":[{"id":"LPB00653","pathogen":"Mycoplasma pneumoniae","target":"P1"}]},{"id":377,"pmid":31523649,"title":"Rapid Detection of Mycoplasma pneumoniae by Loop-Mediated Isothermal Amplification (LAMP) in Clinical Respiratory Specimens","year":2019,"journal":"Iranian Journal of Public Health","authors":"Maryam Arfaatabar, Narjes Noori Goodarzi, Davoud Afshar, Hamed Memariani, Ghasem Azimi, Ensieh Masoorian, Mohammad Reza Pourmand","doi":"","country":"Iran","institute":"Tehran University of Medical Sciences","deparment":"Department of Pathobiology","abstract":"Background: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP) worldwide, especially among children and debilitated populations. The present study aimed to investigate a loop-mediated isothermal amplification (LAMP) technique for rapid detection of M. pneumoniae in clinical specimens collected from patients with pneumonia.\n\nMethods: Throat swabs were collected from 110 outpatients who suffered from pneumonia. Throat swab samples were obtained from patients referred to the hospital outpatient clinics of Tehran University hospitals, Iran in 2017. The presence of M. pneumoniae in the clinical specimens was evaluated by LAMP, PCR and culture methods. Sensitivity and specificity of the LAMP and PCR assays were also determined.\n\nResults: Out of 110 specimens, LAMP assay detected M. pneumoniae in 35 specimens. Detection limit of the LAMP assay was determined to be 33fg \/\u03bcL or \u223c 40 genome copies\/reaction. Moreover, no cross-reaction with genomic DNA from other bacteria was observed. Only 25 specimens were positive by the culture method. The congruence between LAMP assay and culture method was 'substantial' (\u03f0=0.77). Specificity and sensitivity of LAMP assay were 88.2%, 100% in compare with culture. However, the congruence between LAMP assay and PCR assay was 'almost perfect' (\u03f0=0.86). Specificity and sensitivity of LAMP assay were 92.5%, 100% in compare with PCR.\n\nConclusion: Overall, the LAMP assay is a rapid and cost-efficient laboratory test in comparison to other methods including PCR and culture. Therefore, the LAMP method can be applied in identification of M. pneumoniae isolates in respiratory specimens.","lamp_id":[{"id":"LPB00654","pathogen":"Mycoplasma pneumoniae","target":"gyrB"}]},{"id":378,"pmid":29046666,"title":"Molecular Serotype-Specific Identification of Non-type b Haemophilus influenzae by Loop-Mediated Isothermal Amplification","year":2017,"journal":"Frontiers in Microbiology","authors":"Chika Takano, Mitsuko Seki, Dong Wook Kim, Paul E Kilgore, Kazumasa Fuwa, Koji Takahashi, Toshiaki Inazaki, Satoshi Hayakawa","doi":"10.3389\/fmicb.2017.01877","country":"Japan","institute":"Nihon University School of Medicine","deparment":"Department of Pathology and Microbiology","abstract":"Over the past four decades, the incidence of meningitis caused by Haemophilus influenzae in children has decreased due to widespread vaccination against H. influenzae type b (Hib). The incidence of invasive diseases due to H. influenzae types not included in the vaccines, however, has increased. At present, there are a limited number of diagnostics available to detect non-type b H. influenzae. To address this issue, we developed a rapid, simple, and cost-effective method for detecting serotypes of H. influenzae. We designed LAMP primer sets based on published sequences for H. influenzae capsular types a, c, d, e, and f. The assay was evaluated to determine test reactivity, specificity, and sensitivity. To support its use in patients with suspected meningitis, we evaluated the detection limit of the non-Hib serotype specific LAMP assay using bacterial genomic DNA-spiked cerebrospinal fluid (CSF) specimens. The reactivity and specificity of the LAMP assays were confirmed using six serotypes and non-typeable H. influenzae strains, plus eight strains of other Haemophilus species and non-Haemophilus genera. The detection limits of the LAMP assay for capsular types a, c, d, e, and f were 10^2, 10^2, 10^2, 10^3, and 10 copies per reaction, while those of the PCR assay were 10^4, 10^4, 10^3, 10^3, and 10^4 genome copies per reaction, respectively. Using DNA-spiked CSF specimens, the detection limit of the LAMP assay was equivalent to that using purified DNA as the template. However, the detection limit of the PCR was reduced from 10^3 to 10^4 genome copies per reaction for serotype d and from 10^3 to 10^5 genome copies per reaction for serotype e. To the best of our knowledge, this is the first report of a serotype-specific identification assay for H. influenzae using the LAMP method. Our results suggest the potential of LAMP methods for patients with suspected meningitis in resource-limited laboratories or public health surveillance systems.","lamp_id":[{"id":"LPB00655","pathogen":"Haemophilus influenzae","target":"acs1"},{"id":"LPB00656","pathogen":"Haemophilus influenzae","target":"Ccs1, Ccs2, Ccs3, Ccs4-like"},{"id":"LPB00657","pathogen":"Haemophilus influenzae","target":null},{"id":"LPB00658","pathogen":"Haemophilus influenzae","target":"Capsule synthesis"},{"id":"LPB00659","pathogen":"Haemophilus influenzae","target":null}]},{"id":379,"pmid":30529128,"title":"Rapid and Sensitive Detection of Azole-Resistant Aspergillus fumigatus by Tandem Repeat Loop-Mediated Isothermal Amplification","year":2018,"journal":"The Journal of Molecular Diagnostics","authors":"Ling-Shan Yu, Jesus Rodriguez-Manzano, Kenny Malpartida-Cardenas, Thomas Sewell, Oliver Bader, Darius Armstrong-Jame,  Matthew C. Fisher, Pantelis Georgiou","doi":"10.1016\/j.jmoldx.2018.10.004","country":"UK","institute":"Institute of Biomedical Engineering","deparment":"Department of Electrical and Electronic Engineering","abstract":"Invasive fungal infections caused by multiazole-resistant Aspergillus fumigatus are associated with increasing rates of mortality in susceptible patients. Current methods of diagnosing infections caused by multiazole-resistant A. fumigatus are, however, not well suited for use in clinical point-of-care testing or in the field. Loop-mediated isothermal amplification (LAMP) is a widely used method of nucleic acid amplification with rapid and easy-to-use features, making it suitable for use in different resource settings. Here, we developed a LAMP assay to detect a 34 bp tandem repeat, named TR34-LAMP. TR34 is a high-prevalence allele that, in conjunction with the L98H single-nucleotide polymorphism, is associated with the occurrence of multiazole resistance in A. fumigatus in the environment and in patients. This process was validated with both synthetic double-stranded DNA and genomic DNA prepared from azole-resistant isolates of A. fumigatus. Use of our assay resulted in rapid and specific identification of the TR34 allele with high sensitivity, detecting down to 10 genomic copies per reaction within 25 minutes. Fluorescent and colorimetric detections were used for the analysis of 11 clinical isolates as cross validation. These results show that the TR34-LAMP assay has the potential to accelerate the screening of clinical and environmental A. fumigatus to provide a rapid and accurate diagnosis of azole resistance, which current methods struggle to achieve.","lamp_id":[{"id":"LPB00660","pathogen":"Aspergillus fumigatus","target":"cyp51A"}]},{"id":380,"pmid":32151124,"title":"Optimization of isothermal amplification method for Mycobacterium tuberculosis detection and visualization method for fieldwork","year":2020,"journal":"Turkish Journal of Medical Sciences","authors":"Hatice Esra A\u011fel, Hasan Sa\u011fcan, Ismail Ceyhan, Riza Durmaz","doi":"10.3906\/sag-1910-6","country":"Turkey","institute":"T\u00dcB\u0130TAK Marmara Research Center","deparment":"Materials Institute","abstract":"Background\/aim: Tuberculosis is still one of the most contagious diseases around the world. Key factors of tuberculosis control are rapid diagnostic, efficient treatment, and prevention of contamination by surveillance and monitoring. However, culture is the gold standard method for laboratory diagnosis of tuberculosis; the results are several weeks to obtain. In order to prevent contamination of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. The aim of this study is to optimize the loop-mediated isothermal amplification (LAMP) method, which provides a much faster and more sensitive result than the polymerase chain reaction (PCR) method and allows the replication of target nucleic acid sequences under isothermal conditions without the need for laboratory infrastructure.\n\nMaterials and methods: Sputum samples were homogenized with 5% trypsin solution in CaCl2 to obtain DNA.DNA was purified using QIAGEN QIAamp DNA mini kit. LAMP primers were design using Primer explorer V5 program according to IS6110 gene of Mycobacterium tuberculosis. NEB Bst 3.0 DNA polymerase kit was used for LAMP reactions. Besides, LAMP reactions were compared with TaqMan based RT-PCR method using NEB\u2019s Taq polymerase kit. Finally, for visualization of LAMP products, lateral flow dipsticks that produced by Milenia Biotec, colorimetric 2X LAMP master mix that produced by NEB and 2% w\/v agarose gel electrophoresis methods were used.\n\nResults: Optimum amplification temperature for LAMP was found to be 71.4 \u00b0C. The detection limit of the method was 100 CFU\/mL and sensitivity was determined 100% compared to five different Mycobacterium species.\n\nConclusion: The current study indicated that the LAMP-LFD and colorimetric LAMP protocol optimized with sputum samples can be reliable used as a rapid, sensitive and specific assay in the diagnosis of tuberculosis in the field.","lamp_id":[{"id":"LPB00661","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"}]},{"id":381,"pmid":29594577,"title":"Detection of nucleic acids and elimination of carryover contamination by using loop-mediated isothermal amplification and antarctic thermal sensitive uracil-DNA-glycosylase in a lateral flow biosensor: application to the detection of Streptococcus pneumoniae","year":2018,"journal":"Microchimica Acta","authors":"Yi Wang, Yan Wang, Dongxun Li, Jianguo Xu, Changyun Ye","doi":"10.1007\/s00604-018-2723-8","country":"","institute":"","deparment":"","abstract":"The authors report on a loop-mediated isothermal amplification (LAMP) scheme that uses antarctic thermally sensitive uracil-DNA-glycosylase (AUDG) for simultaneous detection of nucleic acids and elimination of carryover contamination. It was applied in a lateral flow assay (LFA) format. The assay has attractive features in that it does not require the use of labeled primers or probes, and can eliminate false-positive results generated by unwanted hybridization between two labeled primers or between a labeled primer and probe. LAMP amplification and AUDG digestion are conducted in a single pot, and the application of a closed-tube reaction prevents false-positives due to carryover contamination. The method was applied to the detection of the human pathogen Streptococcus pneumoniaein in pure cultures and spiked blood samples. This LFA can detect S. pneumoniae in pure cultures with a 25 fg.\u03bcL-1 detection limit and in spiked blood samples with a 470 cfu.mL-1 detection limit. Conceivably, this assay can be applied to the detection of various other targets if the specific LAMP primers are available. ","lamp_id":[{"id":"LPB00662","pathogen":"Streptococcus pneumoniae","target":"ply"}]},{"id":382,"pmid":35111144,"title":"Development of Loop-Mediated Isothermal Amplification Assay Targeting lytA and psaA Genes for Rapid and Visual Diagnosis of Streptococcus pneumoniae Pneumonia in Children","year":2022,"journal":"Frontiers in Microbiology","authors":"Shuheng Du, Chao Yan, Bing Du, Hanqing Zhao, Guanhua Xue, Ping Zheng, Yanling Feng, Jinghua Cui, Lin Gan, Junxia Feng, Zheng Fan, Ziying Xu, Tongtong Fu, Hanyu Fu, Qun Zhang, Nannan Li, Rui Zhang, Shiyu Liu, Xiaoran Li, Xiaohu Cui, Yao Zhou, Qi Zhang, Yaodong Chen, Jing Yuan","doi":"10.3389\/fmicb.2021.816997","country":"China","institute":"Capital Institute of Pediatrics","deparment":"Department of Bacteriology","abstract":"Streptococcus pneumoniae (S. pneumoniae) is a common major human pathogen associated with community-acquired pneumonia, septicemia, meningitis, and otitis media. It is difficult to isolate and identify S. pneumoniae form clinical samples. To evaluate a novel, rapid, sensitive, and specific loop-mediated isothermal amplification (LAMP) assay to detect S. pneumoniae pneumonia in children, we designed specific LAMP primers targeting lytA and psaA genes. We optimized the reaction time and reaction system, and evaluated its sensitivity and specificity of detection using real-time turbidity monitoring and visual observation. We also analyzed the molecular characteristics of the isolates obtained from the positive samples. The primer sets LytA-1 and PsaA-2 amplified the genes in the shortest times, and 63\u00b0C was confirmed as the optimum reaction temperature. The detection sensitivity of each reaction was 10 and 100 copies\/\u03bcL with primer sets LytA-1 and PsaA-2, respectively. This LAMP assay showed no cross-reactivity with other 27 pathogens. To describe the availability of this method, we collected 748 clinical samples from children with pneumonia. Among them, 135 were confirmed to be S. pneumoniae positive by LAMP. The sensitivity was 100% (95% CI 96.4-100%), specificity 99.0% (95% CI 97.8-99.6%). Including them, 50 were co-infected with Mycoplasma pneumoniae. This LAMP assay detected S. pneumoniae in 1 h and the results can be identified with visual naked eyes. Thus, it will be a powerful tool for S. pneumoniae early diagnosis and effective antibiotic therapy.","lamp_id":[{"id":"LPB00663","pathogen":"Streptococcus pneumoniae R6","target":"lytA"},{"id":"LPB00664","pathogen":"Streptococcus pneumoniae R6","target":"psaA"}]},{"id":383,"pmid":30916220,"title":"Rapid, specific, and sensitive detection of the ureR_1 gene in Klebsiella pneumoniae by loop-mediated isothermal amplification method","year":2019,"journal":"Brazilian Journal of Medical and Biological Research","authors":"Chao Li, Gongyu Fu, Yaoqiang Shi, A-Mei Zhang, Xueshan Xia, Yue Fang, Xiaoqin Mao, Jie Jiang, Yuzhu Song, Guangying Yang","doi":"10.1590\/1414-431X20198186","country":"China","institute":"Kunming University of Science and Technology","deparment":"Faculty of Life Science and Technology","abstract":"Klebsiella pneumoniae is one of the main pathogenic bacteria that causes nosocomial infections, such as pneumonia, urinary tract infection, and sepsis. Therefore, the rapid and accurate detection of K. pneumoniae is important for the timely treatment of infectious patients. This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of K. pneumoniae-specific gene ureR_1 (Gene ID: 11847803). The ureR_1 gene was obtained through local and online BLAST, and the specific primers were designed for its detection. Positive reactions were observed on all 140 K. pneumoniae clinical isolates while all the 82 non-K. pneumoniae clinical isolates were negative. Plasmids with the specific gene and the mouse blood with K. pneumoniae were used for sensitivity analysis. The detection limit of the LAMP was 1 bacterium\/reaction. The results showed that the LAMP targeted to ureR_1 is a fast, specific, sensitive, inexpensive, and suitable method for the detection of K. pneumoniae.","lamp_id":[{"id":"LPB00665","pathogen":"Klebsiella pneumoniae subsp. pneumoniae HS11286","target":"ureR_1"}]},{"id":384,"pmid":30915049,"title":"Rapid and Specific Detection of All Known Nipah virus Strains' Sequences With Reverse Transcription-Loop-Mediated Isothermal Amplification","year":2019,"journal":"Frontiers in Microbiology","authors":"Liping Ma, Zhen Chen, Wuxiang Guan, Quanjiao Chen, Di Liu","doi":"10.3389\/fmicb.2019.00418","country":"China","institute":"Wuhan Institute of Virology","deparment":"CAS Key Laboratory of Special Pathogens and Biosafety","abstract":"Nipah virus (NiV) is a zoonotic virus and can be transmitted through contaminated food or directly between people. NiV is classified as a Biosafety Level 4 agent, not only because of its relatively high case fatality rate, but also because there is no vaccine or other medical countermeasures and it appears to be transmitted by fomites\/particulates. The development of rapid detection assay for NiV is of great importance because no effective field test is currently available. In this study, an isothermal (65\u00b0C) reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method was developed, targeting the nucleocapsid protein (N) gene, for the rapid detection of NiV, and was compared with conventional RT-PCR. Three pseudoviruses of NiV N gene representing all known strains were constructed to replace live NiV. A set of RT-LAMP primers, targeting a highly conserved region of the N gene in the viral genome was designed to identify all known NiV strains. Sensitivity tests indicated that the detection limit of the RT-LAMP assay was approximately 100 pg of total NiV pseudovirus RNA, which is at least 10-fold higher than that of conventional RT-PCR. Specificity tests showed that there was no cross-reactivity with nucleocapsid protein gene of Hendra virus, Newcastle disease virus, Japanese encephalitis virus, or Influenza A virus. The RT-LAMP assay provides results within 45 min, and requires no sophisticated instruments, except an isothermal water bath or metal bath with 1 \u03bcl calcein indicator. An analysis of the clinical samples showed that the assay had good stability. In conclusion, systematic experiments have shown that the RT-LAMP assay developed here effectively detects three NiV pseudoviruses representing all known strains of NiV, with high specificity, sensitivity and stability.","lamp_id":[{"id":"LPB00666","pathogen":"Nipah virus","target":"N"}]},{"id":385,"pmid":30651773,"title":"Establishment of loop-mediated isothermal amplification for rapid detection of Pseudomonas aeruginosa","year":2018,"journal":"Experimental and Therapeutic Medicine","authors":"Chao Li, Yaoqiang Shi, Guangying Yang, Xue-Shan Xia, Xiaoqin Mao, Yue Fang, A-Mei Zhang, Yuzhu Song","doi":"10.3892\/etm.2018.6910","country":"China","institute":"Kunming University of Science and Technology","deparment":"Molecular Diagnosis Laboratory","abstract":"Pseudomonas aeruginosa is one of the three most pathogenic bacteria that frequently cause life-threatening opportunistic human infections, pneumonia, and lower respiratory tract infections in immunocompromised hosts, particularly in the burns ward. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of P. aeruginosa-specific gene hypothetical protein (GenBank ID: 882161). The gene was obtained through local and online BLAST, and specific primers were designed for this gene. Reaction conditions were optimized at 65\u00b0C for 30 min and 80\u00b0C for 2 min, whereas the reaction system contained 5.2 mM Mg2+, 8 U Bst 2.0 DNA polymerase, 1.4 mM deoxyribonucleotide and 0.2 and 1.6 \u00b5M of the outer and inner primers, respectively. The LAMP method was evaluated using 150 P. aeruginosa and 170 non-P. aeruginosa strains. Positive reactions were observed on 150 P. aeruginosa strains, whereas all non-P. aeruginosa strains exhibited negative results. Plasmids with the specific gene and mouse blood with P. aeruginosa were used for sensitivity assay. The detection limit of LAMP was 1 bacterium\/reaction. Results indicated that the LAMP method targeted to hypothetical protein is a fast, specific, sensitive, inexpensive and suitable method for detection of P. aeruginosa.","lamp_id":[{"id":"LPB00667","pathogen":"Pseudomonas aeruginosa","target":"hypothetical protein"}]},{"id":386,"pmid":36169448,"title":"Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2","year":2022,"journal":"Microbiology Spectrum","authors":"Concha Ortiz-Cartagena, Laura Fern\u00e1ndez-Garc\u00eda, Lucia Blasco, Olga Pacios, In\u00e9s Bleriot, Mar\u00eda L\u00f3pez, Rafael Cant\u00f3n, Mar\u00eda Tom\u00e1s","doi":"10.1128\/spectrum.02398-22","country":"Spain","institute":"Biomedical Research Institute A Coru\u00f1a (INIBIC)","deparment":"Translational and Multidisciplinary Microbiology (MicroTM)","abstract":"At the end of 2019, a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused a pandemic that persists to date and has resulted in more than 6.2 million deaths. In the last couple of years, researchers have made great efforts to develop a diagnostic technique that maintains high levels of sensitivity and specificity, since an accurate and early diagnosis is required to minimize the prevalence of SARS-CoV-2 infection. In this context, CRISPR-Cas systems are proposed as promising tools for development as diagnostic techniques due to their high specificity, highlighting that Cas13 endonuclease discriminates single nucleotide changes and displays collateral activity against single-stranded RNA molecules. With the aim of improving the sensitivity of diagnosis, this technology is usually combined with isothermal preamplification reactions (SHERLOCK, DETECTR). Based on this, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP)-CRISPR-Cas13a method for SARS-CoV-2 virus detection in nasopharyngeal samples without using RNA extraction that exhibits 100% specificity and 83% sensitivity, as well as a positive predictive value (PPV) of 100% and negative predictive values (NPVs) of 100%, 81%, 79.1%, and 66.7% for cycle threshold (CT) values of <20, 20 to 30, >30 and overall, respectively.","lamp_id":[{"id":"LPB00668","pathogen":"SARS-CoV-2","target":"N"}]},{"id":387,"pmid":30547031,"title":"rpoB Targeted Loop-Mediated Isothermal Amplification (LAMP) Assay for Consensus Detection of Mycobacteria Associated With Pulmonary Infections","year":2018,"journal":"Frontiers in Medicine","authors":"Simon Grandjean Lapierre, Michel Drancourt","doi":"10.3389\/fmed.2018.00332","country":"","institute":"","deparment":"","abstract":"Loop-mediated isothermal amplification (LAMP) is a nucleic acid method which has been used to identify mycobacteria including Mycobacterium tuberculosis in clinical microbiology laboratory and point of care settings. Previously published LAMP protocols for detection of mycobacterial species used conventional specific primer and targeted the 16S rRNA, gyrB, and insertion sequence genes. We developed and evaluated a LAMP assay targeting a mycobacterial rpoB gene conserved sequence and incorporating degenerate primers. This assay allowed consensus detection of mycobacterial species from pure culture, clinical respiratory tract samples, and mycobacteria growth indicator tube (MGIT) liquid-based culture medium. A panel of twenty mycobacterial species were successfully detected at detection thresholds of 10^2 CFU\/mL and 10^3 CFU\/mL when respectively performed on pure culture suspension or sputum and MGIT broth. The inclusion of degenerate bases in LAMP primers increased the diversity of mycobacterial species identified by the assay without negatively affecting analytical sensitivity. LAMP-based consensus detection of multiple pathogens can be achieved with degenerate primers therefore allowing the design of rapid multi-disease screening assays. Despite high analytical sensitivity, species specificity and the advantageous operational characteristics of LAMP over PCR, challenges such as potential ambiguity in visual interpretation of results and occasional non-specific amplification precludes the implementation of novel LAMP assay in routine diagnostics both in centralized and point-of-care laboratory.","lamp_id":[{"id":"LPB00669","pathogen":"Mycobacterium tuberculosis H37Rv","target":"rpoB"}]},{"id":388,"pmid":30636048,"title":"A loop-mediated isothermal amplification assay for the diagnosis of pulmonary tuberculosis","year":2019,"journal":"Letters in Applied Microbiology","authors":"G Sharma, R Tewari, S K Dhatwalia, R Yadav, D Behera, S Sethi","doi":"10.1111\/lam.13115","country":"","institute":"","deparment":"","abstract":"Quantitated Mycobacterium tuberculosis (M.tb) H37Rv DNA was used to analyse the sensitivity and the specificity was assessed using DNA isolated from the reference strain H37Rv, 12 nontuberculous mycobacterium (NTM) species and five nonmycobacterium species. Furthermore, performance of the assay was evaluated on the sputum samples and compared with smear microscopy, culture and PCR. mpt64 (also called mpb64 or Rv1980c) loop-mediated isothermal amplification (LAMP) successfully detected 1 pg DNA within 40 min and successfully rejected NTMs and other bacterial species tested. It specifically detected all the 119 confirmed TB cases and 100 of the 104 control cases. The resulting sensitivity and specificity of LAMP assay was found to be 100% (95% CI: 96\u00b779-100%) and 96\u00b715% (95% CI; 90\u00b744-98\u00b794%) respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Loop-mediated isothermal amplification (LAMP) is a technique for isothermal DNA amplification suitable for cost-limited settings as it prevents the use of sophisticated instruments. Using mpt64 antigenic protein gene, we developed a LAMP assay especially for organisms of the M. tuberculosis complex. mpt64 LAMP assay showed 100% sensitivity and detected all the bacteriologically and clinically positive TB cases not detected by smear, culture or PCR methods.","lamp_id":[{"id":"LPB00670","pathogen":"Mycobacterium tuberculosis","target":"mpt64"}]},{"id":389,"pmid":21883320,"title":"Comparison of loop-mediated isothermal amplification and real-time PCR for the diagnosis of tuberculous pleurisy","year":2011,"journal":"Letters in Applied Microbiology","authors":"B Yang, X Wang, H Li, G Li, Z Cao, X Cheng","doi":"10.1111\/j.1472-765X.2011.03141.x","country":"","institute":"","deparment":"","abstract":"Aims: Tuberculous pleurisy is an important cause of pleural effusions in areas with a high incidence of tuberculosis. In this study, we developed an IS1081-based LAMP for the detection of Mycobacterium tuberculosis complex and investigated its usefulness in the diagnosis of tuberculous pleurisy.\n\nMethods and results: Investigation of pleural effusion samples from patients with tuberculous pleurisy, majority of them smear-\/culture-negative, and control individuals with non-TB diseases showed that the LAMP assay with incubation time of 60 min has much higher specificity and the LAMP assay with incubation time of 90 min has significantly higher sensitivity in the diagnosis of tuberculous pleurisy, as compared with fluorescent real-time PCR.\n\nConclusions: The MTBC-LAMP is a useful assay for the diagnosis of tuberculous pleurisy, especially in pleural effusion smear-\/culture-negative patients.\n\nSignificance and impact of the study: Tuberculous pleural effusion usually contains low number of mycobacteria, which leads to low diagnostic sensitivity of acid-fast staining and mycobacterial culture methods. In this study, we developed a simple and sensitive LAMP assay for the diagnosis of tuberculous pleurisy. This assay should have broad application in resource-limited settings.","lamp_id":[{"id":"LPB00671","pathogen":"Mycobacterium tuberculosis","target":"IS1081"}]},{"id":390,"pmid":28811193,"title":"A novel detection procedure for mutations in the 23S rRNA gene of Mycoplasma pneumoniae with peptide nucleic acid-mediated loop-mediated isothermal amplification assay","year":2017,"journal":"Journal of Microbiological Methods","authors":"Jun Sakai, Takuya Maeda, Norihito Tarumoto, Kazuhisa Misawa, Shinsuke Tamura, Kazuo Imai, Toshiyuki Yamaguchi, Satoshi Iwata, Takashi Murakami, Shigefumi Maesaki","doi":"10.1016\/j.mimet.2017.08.009","country":"","institute":"","deparment":"","abstract":"Rapid and easy detection of a single nucleotide point mutation of bacterial genes, which is directly linked to drug susceptibility, is essential for the proper use of antimicrobial agents. Here, we established a detection method using a peptide nucleic acid mediated loop-mediated amplification (LAMP) assay for macrolide (ML)-susceptible Mycoplasma pneumoniae. This assay specifically detected the absence of missense mutations encoding the central loop of domain V in the gene encoding 23S rRNA, which can reduce the affinity for MLs and subsequently generate ML-resistant strains of M. pneumoniae. Reactions were performed at 62\u00b0C for 60min and targeted gene amplifications were detected by real-time turbidity with a turbidimeter and naked-eye inspection of a color change. The assay had an equivalent detection limit of 100.0fg of DNA with the turbidimeter and showed specificity against 54 types of pathogens, whereas amplification was completely blocked, even at 100.0pg of DNA per reaction, in the presence of point mutations at 2063A and 2064A. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. This method would be a simple and rapid protocol for single nucleotide polymorphism genotyping as point-of-care testing technology without amplification of the sequences carrying the point mutations 2063A and 2064A in ML-resistant M. pneumoniae strains.","lamp_id":[{"id":"LPB00672","pathogen":"Mycoplasma pneumoniae","target":"23S rRNA"}]},{"id":391,"pmid":33493196,"title":"Development of a loop-mediated isothermal amplification (LAMP) method for specific detection of Mycobacterium bovis","year":2021,"journal":"PLOS Neglected Tropical Diseases","authors":"Thoko Flav Kapalamula, Jeewan Thapa, Mwangala Lonah Akapelwa, Kyoko Hayashida, Stephen V Gordon, Bernard Mudenda Hang' Ombe, Musso Munyeme, Eddie Samuneti Solo, Precious Bwalya, Mirriam Ethel Nyenje, Aki Tamaru, Yasuhiko Suzuki, Chie Nakajima","doi":"10.1371\/journal.pntd.0008996","country":"Japan","institute":"Research Center for Zoonosis Control","deparment":"Division of Bioresources","abstract":"Bovine tuberculosis (TB) caused by Mycobacterium bovis is a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and accurate detection of M. bovis is fundamental for controlling the disease in animals and humans, and for the proper treatment of patients as one of the first-line anti-TB drug, pyrazinamide, is ineffective against M. bovis. Currently, there are no rapid, simplified and low-cost diagnostic methods that can be easily integrated for use in many developing countries. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for specific identification of M. bovis by targeting the region of difference 4 (RD4), a 12.7 kb genomic region that is deleted solely in M. bovis. The assay's specificity was evaluated using 139 isolates comprising 65 M. bovis isolates, 40 M. tuberculosis isolates, seven M. tuberculosis complex reference strains, 22 non-tuberculous mycobacteria and five other bacteria. The established LAMP detected only M. bovis isolates as positive and no false positives were observed using the other mycobacteria and non-mycobacteria tested. Our LAMP assay detected as low as 10 copies of M. bovis genomic DNA within 40 minutes. The procedure of LAMP is simple with an incubation at a constant temperature. Results are observed with the naked eye by a color change, and there is no need for expensive equipment. The established LAMP can be used for the detection of M. bovis infections in cattle and humans in resource-limited areas.","lamp_id":[{"id":"LPB00673","pathogen":"Mycobacterium tuberculosis AF2122\/97","target":"RD4 deletion"}]},{"id":392,"pmid":33406154,"title":"Development and evaluation of a multiplex loop-mediated isothermal amplification (LAMP) assay for differentiation of Mycobacterium tuberculosis and non-tuberculosis mycobacterium in clinical samples","year":2021,"journal":"PLoS One","authors":"Jeeyong Kim, Borae G Park, Da Hye Lim, Woong Sik Jang, Jeonghun Nam, Do-CiC Mihn, Chae Seung Lim","doi":"10.1371\/journal.pone.0244753","country":"Republic of Korea.","institute":"Korea University Guro Hospital","deparment":"Department of Laboratory Medicine","abstract":"Introduction: The rapid and accurate diagnosis of tuberculosis (TB) is important to reduce morbidity and mortality rates and risk of transmission. Therefore, molecular detection methods such as a real-time PCR-based assay for Mycobacterium tuberculosis (MTB) have been commonly used for diagnosis of TB. Loop-mediated isothermal amplification (LAMP) assay was believed to be a simple, quick, and cost-effective isothermal nucleic acid amplification diagnostic test for infectious diseases. In this study, we designed an in-house multiplex LAMP assay for the differential detection of MTB and non-tuberculosis mycobacterium (NTM), and evaluated the assay using clinical samples.\n\nMaterial and methods: For the multiplex LAMP assay, two sets of specific primers were designed: the first one was specific for IS6110 genes of MTB, and the second one was universal for rpoB genes of mycobacterium species including NTM. MTB was confirmed with a positive reaction with both primer sets, and NTM was identified with a positive reaction by only the second primer set without a MTB-specific reaction. Total 333 clinical samples were analyzed to evaluate the multiplex LAMP assay. Clinical samples were composed of 195 positive samples (72 MTB and 123NTM) and 138 negative samples. All samples were confirmed positivity or negativity by real-time PCR for MTB and NTM. Analytical sensitivity and specificity were evaluated for the multiplex LAMP assay in comparison with acid fast bacilli staining and the culture method.\n\nResults: Of 123 NTM samples, 121 were identified as NTM and 72\/72 MTB were identified as MTB by the multiplex LAMP assay. False negative reactions were seen only in two NTM positive samples with co-infection of Candida spp. All 138 negative samples were identified as negative for MTB and NTM. Analytical sensitivity of the multiplex LAMP assay was 100% (72\/72) for MTB, and 98.4% (121\/123) for NTM. And the specificity of assay was 100% (138\/138) for all.\n\nConclusions: Our newly designed multiplex LAMP assay for MTB and NTM showed relatively good sensitivity in comparison with previously published data to detect isolated MTB. This multiplex LAMP assay is expected to become a useful tool for detecting and differentiating MTB from NTM rapidly at an acceptable sensitivity.","lamp_id":[{"id":"LPB00674","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"},{"id":"LPB00675","pathogen":"Mycobacterium","target":"rpoB"}]},{"id":393,"pmid":34408738,"title":"Rapid and Visual Differentiation of Mycobacterium tuberculosis From the Mycobacterium tuberculosis Complex Using Multiplex Loop-Mediated Isothermal Amplification Coupled With a Nanoparticle-Based Lateral Flow Biosensor","year":2021,"journal":"Frontiers in Microbiology","authors":"Xinggui Yang, Junfei Huang, Xu Chen, Ziyu Xiao, Xiaojuan Wang, Yijiang Chen, Wenlin Zheng, Wei Chen, Huijuan Chen, Shiguang Lei, Yong Hu, Shijun Li","doi":"10.3389\/fmicb.2021.708658","country":"China","institute":"Guizhou Medical University","deparment":"Public Health School","abstract":"Tuberculosis (TB) is a chronic infectious disease mainly caused by Mycobacterium tuberculosis (MTB), but other members of the Mycobacterium tuberculosis complex (MTBC), especially Mycobacterium bovis (pyrazinamide-resistant organisms), may also be involved. Thus, the ability to rapidly detect and identify MTB from other MTBC members (e.g., M. bovis, Mycobacterium microti, Mycobacterium africanum) is essential for the prevention and treatment of TB. A novel diagnostic method for the rapid detection and differentiation of MTB, which employs multiplex loop-mediated isothermal amplification (mLAMP) combined with a nanoparticle-based lateral flow biosensor (LFB), was established (mLAMP-LFB). Two sets of specific primers that target the IS6110 and mtp40 genes were designed according to the principle of LAMP. Various pathogens were used to optimize and evaluate the mLAMP-LFB assay. The optimal conditions for mLAMP-LFB were determined to be 66\u00b0C and 40 min, and the amplicons were directly verified by observing the test lines on the biosensor. The LAMP assay limit of detection (LoD) was 125 fg per vessel for the pure genomic DNA of MTB and 4.8 \u00d7 10^3 CFU\/ml for the sputum samples, and the analytical specificity was 100%. In addition, the whole process, including the clinical specimen processing (35 min), isothermal amplification (40 min), and result confirmation (1-2 min), could be completed in approximately 80 min. Thus, mLAMP-LFB is a rapid, reliable, and sensitive method that is able to detect representative members of MTBC and simultaneously differentiate MTB from other MTBC members, and it can be used as a potential screening tool for TB in clinical, field, and basic laboratory settings.","lamp_id":[{"id":"LPB00677","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"},{"id":"LPB00678","pathogen":"Mycobacterium tuberculosis","target":"mtp40"}]},{"id":394,"pmid":33987108,"title":"Development and Preliminary Application of Multiplex Loop-Mediated Isothermal Amplification Coupled With Lateral Flow Biosensor for Detection of Mycobacterium tuberculosis Complex","year":2021,"journal":"Frontiers in Cellular and Infection Microbiology","authors":"Xingyun Wang, Guirong Wang, Yacui Wang, Shuting Quan, Hui Qi, Lin Sun, Chen Shen, Hairong Huang, Weiwei Jiao, Adong Shen","doi":"10.3389\/fcimb.2021.666492","country":"","institute":"","deparment":"","abstract":"The aim of this study was to develop a simple and reliable method to detect Mycobacterium tuberculosis complex (MTBC) and verify its clinical application preliminarily. A loop-mediated isothermal amplification method coupled with lateral flow biosensor (LAMP-LFB) assay, was developed and evaluated for detection of MTBC. Two sets of primers, which targeted IS6110 and IS1081 sequences of MTBC, were designed for establishment of multiplex LAMP-LFB assay. The amplicons were labelled with biotin and fluorescein isothiocyanate (FITC) by adding FITC labelled primer and biotin-14-dATP and biotin-14-dCTP and could be visualized using LFB. The optimal reaction conditions of multiplex LAMP-LFB assay confirmed were 66\u00b0C for 50 min. The analytical sensitivity of multiplex LAMP-LFB is 10 fg of genomic templates using pure culture, and no cross-reactivity with other common bacteria and non-tuberculous mycobacteria strains was obtained. A total of 143 clinical samples collected from 100 TB patients (62 definite TB cases and 38 probable TB cases) and 43 non-TB patients were used for evaluating the feasibility of multiplex LAMP-LFB assay. The multiplex LAMP-LFB (82.0%, 82\/100) showed higher sensitivity than culture (47.0%, 47\/100, P < 0.001) and Xpert MTB\/RIF (54.0%, 54\/100, P < 0.001). Importantly, the multiplex LAMP-LFB assay detected additional 28 probable TB cases, which increased the percentage of definite TB cases from 62.0% (62\/100) to 90.0% (90\/100). The specificity of multiplex LAMP-LFB assay in patients without TB was 97.7% (42\/43). Therefore, multiplex LAMP-LFB assay is a simple, reliable, and sensitive method for MTBC detection, especially in probable TB cases and resource limited settings.","lamp_id":[{"id":"LPB00679","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"},{"id":"LPB00680","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS1081"}]},{"id":395,"pmid":34176103,"title":"Highly specific and sensitive detection of the Mycobacterium tuberculosis complex using multiplex loop-mediated isothermal amplification combined with a nanoparticle-based lateral flow biosensor","year":2021,"journal":"Brazilian Journal of Microbiology","authors":"Xu Chen, Junfei Huang, Ziyu Xiao, Xingui Yang, Yijiang Chen, Wenlin Zheng, Wei Chen, Huijuan Chen, Shijun Li","doi":"10.1007\/s42770-021-00520-4","country":"","institute":"","deparment":"","abstract":"Tuberculosis (TB) is the deadliest infectious caused by Mycobacterium tuberculosis complex (MTBC). Because most TB cases occur within low-income populations, developing a specific, sensitive, cost-saving, and rapid point-of-care test for the early diagnosis of TB is important for achieving the WHO's End Tuberculosis Strategy. In the current study, a novel nucleic acid detection strategy that includes multiplex loop-mediated isothermal amplification combined with a nanoparticle-based lateral flow biosensor (mLAMP-LFB) was used to detect MTBC. The two sets of LAMP primers specific to the IS6110 and gyrB genes of MTBC were successfully designed and validated for the detection of MTBC. The preferred reaction conditions for this assay were confirmed to be 65 \u00b0C for 40 min, and the amplification products could be visually identified through LFB within 2 min. The full assay process, including genomic DNA template extraction, LAMP reaction, and product detection, could be completed in 80 min. The limit detection of the assay was 100 fg of DNA in pure culture. The specificity of the assay was 100%, and it had no cross-reactions to other strains. Thus, the m-LAMP-LFB technology established in the present study was an objective, rapid, simple, and sensitive assay for MTBC identification, which could be applied in a clinical setting, especially in resource-constrained regions of the world.","lamp_id":[{"id":"LPB00681","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"},{"id":"LPB00682","pathogen":"Mycobacterium tuberculosis H37Rv","target":"gyrB"}]},{"id":396,"pmid":35715135,"title":"Electromagnetically-driven integrated microfluidic platform using reverse transcription loop-mediated isothermal amplification for detection of severe acute respiratory syndrome coronavirus 2","year":2022,"journal":"Analytica Chimica Acta","authors":"Yu-Shiuan Tsai, Chih-Hung Wang, Huey-Pin Tsai, Yan-Shen Shan, Gwo-Bin Lee","doi":"10.1016\/j.aca.2022.340036","country":"","institute":"","deparment":"","abstract":"Rapid, sensitive and accurate diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of great need for effective quarantining and treatment. Real-time reverse-transcription polymerase chain reaction requiring thermocyling has been commonly used for diagnosis of SARS-CoV-2 though it may take two to 4 h before lengthy sample pretreatment process and require bulky apparatus and well-trained personnel. Since multiple reverse transcription loop-mediated isothermal amplification (multiple RT-LAMP) process without thermocycling is sensitive, specific and fast, an electromagnetically-driven microfluidic chip (EMC) was developed herein to lyse SARS-CoV-2 viruses, extract their RNAs, and perform qualitative analysis of three marker genes by on-chip multiple RT-LAMP in an automatic format within 82 min at a limit of detection of only \u223c5000 copies per reaction (i.e. 200 virus\/ \u03bcL). This compact EMC may be especially promising for SARS-CoV-2 diagnostics in resource-limited countries.","lamp_id":[{"id":"LPB00683","pathogen":"SARS-CoV-2","target":"RdRp"},{"id":"LPB00684","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00685","pathogen":"SARS-CoV-2","target":"N"}]},{"id":397,"pmid":27833606,"title":"Surveillance of Dihydropteroate Synthase Genes in Stenotrophomonas maltophilia by LAMP: Implications for Infection Control and Initial Therapy","year":2016,"journal":"Frontiers in Microbiology","authors":"Jin Zhao, Yubin Xing, Wei Liu, Wentao Ni, Chuanqi Wei, Rui Wang, Yunxi Liu, Youning Liu","doi":"10.3389\/fmicb.2016.01723","country":"","institute":"","deparment":"","abstract":"Stenotrophomonas maltophilia is a common nosocomial pathogen that causes high morbidity and mortality. Because of its inherent extended antibiotic resistance, therapeutic options for S. maltophilia are limited, and sulfamethoxazole\/trimethoprim (SXT) is the only first-line antimicrobial recommended. However, with the spread of dihydropteroate synthase (sul1 and sul2) genes, global emergence of SXT resistance has been reported. There is an urgent need to develop a rapid and sensitive but cost-efficient method to monitor the dissemination of sul genes. In this study, we developed loop-mediated isothermal amplification (LAMP) assays for sul1 and sul2 using real-time turbidity and hydroxy naphthol blue coloration methods. The assays could quickly detect sul genes with high sensitivity and specificity. The LAMP detection limit was 0.74 pg\/reaction of extracted genomic DNA for sul1 and 2.6 pg\/reaction for sul2, which were both 10-fold more sensitive than the corresponding traditional PCR assays. Additionally, the LAMP assays could positively amplify DNA from sul1-producing strains, but not from the negative controls. We then used the LAMP assays to investigate the dissemination of sul genes among S. maltophilia isolates from patients in three hospitals in Beijing, China. Among 450 non-duplicated samples collected during 2012-2014, 56 (12.4%) strains were SXT-resistant. All these SXT-resistant strains were positive for sul genes, with 35 (62.5%) carrying sul1, 17 (30.4%) carrying sul2, and 4 (7.1%) carrying both sul1 and sul2, which indicated that sul genes were the predominant resistance mechanism. Of 394 SXT-susceptible strains, 16 were also sul-positive. To provide epidemiological data for the appropriate choice of antimicrobials for treatment of sul-positive S. maltophilia, we further tested the susceptibility to 18 antimicrobials. Among these, sul-positive strains showed the highest susceptibility to tetracycline derivatives, especially minocycline (MIC50\/MIC90, 0.5\/4; susceptibility rate, 95.4%). Ticarcillin-clavulanate and new fluoroquinolones (moxifloxacin and levofloxacin) also showed some in vitro activity. Apart from these three kinds of antimicrobials, other agents showed poor activity against sul-positive strains.","lamp_id":[{"id":"LPB00686","pathogen":"Stenotrophomonas maltophilia","target":"sul1"},{"id":"LPB00687","pathogen":"Stenotrophomonas maltophilia","target":"sul2"}]},{"id":398,"pmid":26985584,"title":"Point-of-care and visual detection of P. aeruginosa and its toxin genes by multiple LAMP and lateral flow nucleic acid biosensor","year":2016,"journal":"Biosensors and Bioelectronics","authors":"Yuting Chen, Nan Cheng, Yuancong Xu, Kunlun Huang, Yunbo Luo, Wentao Xu","doi":"10.1016\/j.bios.2016.03.006","country":"","institute":"","deparment":"","abstract":"This study describes a simple and sensitive approach for visual and point-of-care detection of P. aeruginosa and its toxin genes based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow nucleic acid biosensor (LFNAB). Differentiation of the internal standard gene ecfX and toxin genes (ExoS and ExoU) in P. aeruginosa was determined using FITC-, hex-and digoxin-modified primers in the mLAMP process. In the presence of biotin-and FITC- (hex-, digoxin-) modified primers and Bst DNA polymerase large fragments, the mLAMP produced numerous biotin- and FITC- (hex-, digoxin-) attached duplex DNA products. The products were detected by LFNAB through dual immunoreactions (anti-biotin antibodies on the gold nanoparticle (Au-NP) and biotin on the duplex, anti-FITC (hex, digoxin) antibodies on the LFNAB test line and FITC (hex, digoxin) on the duplex). The accumulation of Au-NPs produced a characteristic red band, enabling visual detection of P. aeruginosa and its toxin genes without instrumentation. After systematic optimization of LFNAB preparation and detecting conditions, the current approach was capable of detecting concentrations as low as 20 CFU\/mL P. aeruginosa or its toxin genes within 50min without complicated instrument, which is more sensitive than PCR. Therefore, this approach provides a simple, pollution free, sensitive, and low-cost point-of-care test for the detection of P. aeruginosa and its toxin genes.","lamp_id":[{"id":"LPB00688","pathogen":"Pseudomonas aeruginosa","target":"ecfX"},{"id":"LPB00689","pathogen":"Pseudomonas aeruginosa","target":"exoS"},{"id":"LPB00690","pathogen":"Pseudomonas aeruginosa","target":"exoU"}]},{"id":399,"pmid":34840805,"title":"Multiplexed detection of respiratory pathogens with a portable analyzer in a \u201craw-sample-in and answer-out\u201d manner","year":2021,"journal":"Microsystems & Nanoengineering","authors":"Nan Li, Minjie Shen, Jiajia Liu, Li Zhang, Huili Wang, Youchun Xu, Jing Cheng","doi":"10.1038\/s41378-021-00321-7","country":"","institute":"","deparment":"","abstract":"Coronavirus disease 2019 (COVID-19) has emerged, rapidly spread and caused significant morbidity and mortality worldwide. There is an urgent public health need for rapid, sensitive, specific, and on-site diagnostic tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, a fully integrated and portable analyzer was developed to detect SARS-CoV-2 from swab samples based on solid-phase nucleic acid extraction and reverse transcription loop-mediated isothermal amplification (RT-LAMP). The swab can be directly inserted into a cassette for multiplexed detection of respiratory pathogens without pre-preparation. The overall detection process, including swab rinsing, magnetic bead-based nucleic acid extraction, and 8-plex real-time RT-LAMP, can be automatically performed in the cassette within 80 min. The functionality of the cassette was validated by detecting the presence of a SARS-CoV-2 pseudovirus and three other respiratory pathogens, i.e., Klebsiella pneumoniae, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The limit of detection (LoD) for the SARS-CoV-2 pseudovirus was 2.5 copies\/\u03bcL with both primer sets (N gene and ORF1ab gene), and the three bacterial species were successfully detected with an LoD of 2.5 colony-forming units (CFU)\/\u03bcL in 800 \u03bcL of swab rinse. Thus, the analyzer developed in this study has the potential to rapidly detect SARS-CoV-2 and other respiratory pathogens on site in a \"raw-sample-in and answer-out\" manner.","lamp_id":[{"id":"LPB00691","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00692","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00693","pathogen":"Klebsiella pneumoniae","target":null},{"id":"LPB00694","pathogen":"Pseudomonas aeruginosa","target":null},{"id":"LPB00695","pathogen":"Stenotrophomonas maltophilia","target":null}]},{"id":400,"pmid":36354496,"title":"Rapid Detection of Attomolar SARS-CoV-2 Nucleic Acids in All-Dielectric Metasurface Biosensors","year":2022,"journal":"Biosensors","authors":"Masanobu Iwanaga","doi":"10.3390\/bios12110987","country":"","institute":"","deparment":"","abstract":"Worldwide infection due to SARS-CoV-2 revealed that short-time and extremely high-sensitivity detection of nucleic acids is a crucial technique for human beings. Polymerase chain reactions have been mainly used for the SARS-CoV-2 detection over the years. However, an advancement in quantification of the detection and shortening runtime is important for present and future use. Here, we report a rapid detection scheme that is a combination of nucleic acid amplification and a highly efficient fluorescence biosensor, that is, a metasurface biosensor composed of a pair of an all-dielectric metasurface and a microfluidic transparent chip. In the present scheme, we show a series of proof-of-concept experimental results that the metasurface biosensors detected amplicons originating from attomolar SARS-CoV-2 nucleic acids and that the amplification was implemented within 1 h. Furthermore, this detection capability substantially satisfies an official requirement of 100 RNA copies\/140 \u03bcL, which is a criterion for the reliable infection tests.","lamp_id":[{"id":"LPB00696","pathogen":"SARS-CoV-2","target":"N"}]},{"id":401,"pmid":36089609,"title":"Colorimetric and fluorometric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for diagnosis of SARS-CoV-2","year":2022,"journal":"Functional & Integrative Genomics","authors":"Galyah Alhamid, Huseyin Tombuloglu, Dalal Motabagani, Dana Motabagani, Ali A. Rabaan, Kubra Unver, Gabriel Dorado, Ebtesam Al-Suhaimi, Turgay Unver","doi":"10.1007\/s10142-022-00900-5","country":"","institute":"","deparment":"","abstract":"The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused millions of infections and deaths worldwide since it infected humans almost 3 years ago. Improvements of current assays and the development of new rapid tests or to diagnose SARS-CoV-2 are urgent. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and propitious assay, allowing to detect both colorimetric and\/or fluorometric nucleic acid amplifications. This study describes the analytical and clinical evaluation of RT-LAMP assay for detection of SARS-CoV-2, by designing LAMP primers targeting N (nucleocapsid phosphoprotein), RdRp (polyprotein), S (surface glycoprotein), and E (envelope protein) genes. The assay's performance was compared with the gold standard RT-PCR, yielding 94.6% sensitivity and 92.9% specificity. Among the tested primer sets, the ones for S and N genes had the highest analytical sensitivity, showing results in about 20 min. The colorimetric and fluorometric comparisons revealed that the latter is faster than the former. The limit of detection (LoD) of RT-LAMP reaction in both assays is 50 copies\/\u00b5l of the reaction mixture. However, the simple eye-observation advantage of the colorimetric assay (with a color change from yellow to red) serves a promising on-site point-of-care testing method anywhere, including, for instance, laboratory and in-house applications.","lamp_id":[{"id":"LPB00697","pathogen":"SARS-CoV-2","target":"S"}]},{"id":402,"pmid":36140632,"title":"Development and Validation of Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) as a Simple and Rapid Diagnostic Tool for SARS-CoV-2 Detection","year":2022,"journal":"Diagnostics","authors":"Ahmad M. Aldossary, Essam A. Tawfik, Musaad A. Altammami, Azzam A. Alquait, Rayan Y. Booq, Bandar K. Sendy, Mohammed S. Alarawi, Takashi Gojobori, Asmaa M. Altamimi, Taghreed A. Alaifan, Ahmed M. Albarrag, Essam J. Alyamani","doi":"10.3390\/diagnostics12092232","country":"","institute":"","deparment":"","abstract":"Since the COVID-19 pandemic outbreak in the world, many countries have searched for quick diagnostic tools to detect the virus. There are many ways to design diagnostic assays; however, each may have its limitations. A quick, sensitive, specific, and simple approach is essential for highly rapidly transmitted infections, such as SARS-CoV-2. This study aimed to develop a rapid and cost-effective diagnostic tool using a one-step Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) approach. The results were observed using the naked eye within 30\u201360 min using turbidity or colorimetric analysis. The sensitivity, specificity, and lowest limit of detection (LoD) for SARS-CoV-2 RNA against the RT-LAMP assay were assessed. This assay was also verified and validated against commercial quantitative RT-PCR used by health authorities in Saudi Arabia. Furthermore, a quick and direct sampling from the saliva, or buccal cavity, was applied after simple modification, using proteinase K and heating at 98 \u00b0C for 5 min to avoid routine RNA extraction. This rapid single-tube diagnostic tool detected COVID-19 with an accuracy rate of 95% for both genes (ORF1a and N) and an LoD for the ORF1a and N genes as 39 and 25 copies\/reaction, respectively. It can be potentially used as a high-throughput national screening for different respiratory-based infections within the Middle East region, such as the MERS virus or major zoonotic pathogens such as Mycobacterium paratuberculosis and Brucella spp., particularly in remote and rural areas where lab equipment is limited.","lamp_id":[{"id":"LPB00698","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00699","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00700","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00701","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00702","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00703","pathogen":"SARS-CoV-2","target":"N"}]},{"id":403,"pmid":36140431,"title":"Detection of SARS-CoV-2 Using Reverse Transcription Helicase Dependent Amplification and Reverse Transcription Loop-Mediated Amplification Combined with Lateral Flow Assay","year":2022,"journal":"Biomedicines","authors":"Aleksandra Anna Zasada, Ewa Mosiej, Marta Prygiel, Maciej Polak, Karol Wdowiak, Kamila Formi\u0144ska, Robert Zi\u00f3\u0142kowski, Kamil \u017bukowski, Kasper Marchlewicz, Adam Nowi\u0144ski, Julia Nowi\u0144ska, Waldemar Rastawicki, El\u017cbieta Malinowska","doi":"10.3390\/biomedicines10092329","country":"","institute":"","deparment":"","abstract":"Rapid and accurate detection and identification of pathogens in clinical samples is essential for all infection diseases. However, in the case of epidemics, it plays a key role not only in the implementation of effective therapy but also in limiting the spread of the epidemic. In this study, we present the application of two nucleic acid isothermal amplification methods\u2014reverse transcription helicase dependent amplification (RT-HDA) and reverse transcription loop-mediated amplification (RT-LAMP)\u2014combined with lateral flow assay as the tools for the rapid detection of SARS-CoV-2, the etiological agent of COVID-19, which caused the ongoing global pandemic. In order to optimize the RT-had, the LOD was 3 genome copies per reaction for amplification conducted for 10\u201320 min, whereas for RT-LAMP, the LOD was 30\u2013300 genome copies per reaction for a reaction conducted for 40 min. No false-positive results were detected for RT-HDA conducted for 10 to 90 min, but false-positive results occurred when RT-LAMP was conducted for longer than 40 min. We concluded that RT-HDA combined with LFA is more sensitive than RT-LAMP, and it is a good alternative for the development of point-of-care tests for SARS-CoV-2 detection as this method is simple, inexpensive, practical, and does not require qualified personnel to perform the test and interpret its results.","lamp_id":[{"id":"LPB00704","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00705","pathogen":"SARS-CoV-2","target":"E"}]},{"id":404,"pmid":36248705,"title":"Rapid and convenient detection of SARS-CoV-2 using a colorimetric triple-target reverse transcription loop-mediated isothermal amplification method","year":2022,"journal":"PeerJ","authors":"Zhu Yang, Nicole Y. Liu, Zhiwei Zhu, Minmin Xiao, Shuzhi Zhong, Qiqi Xue, Lina Nie, Jinhong Zhao","doi":"10.7717\/peerj.14121","country":"","institute":"","deparment":"","abstract":"Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 poses a significant threat to global public health. Early detection with reliable, fast, and simple assays is crucial to contain the spread of SARS-CoV-2. The real-time reverse transcription-polymerase chain reaction (RT-PCR) assay is currently the gold standard for SARS-CoV-2 detection; however, the reverse transcription loop-mediated isothermal amplification method (RT-LAMP) assay may allow for faster, simpler and cheaper screening of SARS-CoV-2. In this study, the triple-target RT-LAMP assay was first established to simultaneously detect three different target regions (ORF1ab, N and E genes) of SARS-CoV-2. The results revealed that the developed triplex RT-LAMP assay was able to detect down to 11 copies of SARS-CoV-2 RNA per 25 \u00b5L reaction, with greater sensitivity than singleplex or duplex RT-LAMP assays. Moreover, two different indicators, hydroxy naphthol blue (HNB) and cresol red, were studied in the colorimetric RT-LAMP assay; our results suggest that both indicators are suitable for RT-LAMP reactions with an obvious color change. In conclusion, our developed triplex colorimetric RT-LAMP assay may be useful for the screening of COVID-19 cases in limited-resource areas.","lamp_id":[{"id":"LPB00706","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"},{"id":"LPB00707","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00708","pathogen":"SARS-CoV-2","target":"E"}]},{"id":405,"pmid":null,"title":"Development of Loop-mediated Isothermal Amplification (LAMP) Assays Using Five Primers Reduces the False-positive Rate in COVID-19 Diagnosis","year":2022,"journal":"medRxiv","authors":"Galyah Alhamid, Huseyin Tombuloglu, Ebtesam Al-Suhaimi","doi":"10.1101\/2022.10.18.22281181","country":"","institute":"","deparment":"","abstract":"The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is a cheaper and faster testing alternative for detecting SARS-CoV-2. However, high false-positive rate due to misamplification is one of the major limitations. To overcome misamplifications, we developed colorimetric and fluorometric RT-LAMP assays. The assay performances was verified by the gold-standard RT-qPCR technique on 150 clinical samples. Compared to other primer sets with six primers (N, S, and RdRp), E-ID1 primer set, including five primers, performed superbly on both colorimetric and fluorometric assays, yielding sensitivities of 89.5% and 100%, respectively, with a limit of detection of 20 copies\/\u00b5L. The colorimetric RT-LAMP had a specificity of 97.2% and an accuracy of 94.5%, while the fluorometric RT-LAMP obtained 96.9% and 98%, respectively. No misamplification was evident even after 120 minutes, which is crucial for the success of this technique. These findings are important to support the use of RT-LAMP in the healthcare systems in fighting COVID-19.","lamp_id":[{"id":"LPB00709","pathogen":"SARS-CoV-2","target":"E"}]},{"id":406,"pmid":35223985,"title":"Development of Loop-Mediated Isothermal Amplification Rapid Diagnostic Assays for the Detection of Klebsiella pneumoniae and Carbapenemase Genes in Clinical Samples","year":2022,"journal":"Frontiers in Molecular Biosciences","authors":"Aurore C Poirier, Dai Kuang, Bianca S Siedler, Khushboo Borah, Jai W Mehat, Jialin Liu, Cui Tai, Xiaoli Wang, Arnoud H M van Vliet, Wei Ma, David R Jenkins, John Clark, Roberto M La Ragione, Jieming Qu, Johnjoe McFadden","doi":"10.3389\/fmolb.2021.794961","country":"","institute":"","deparment":"","abstract":"Klebsiella pneumoniae is an important pathogenic bacterium commonly associated with human healthcare and community-acquired infections. In recent years, K. pneumoniae has become a significant threat to global public and veterinary health, because of its high rates of antimicrobial resistance (AMR). Early diagnosis of K. pneumoniae infection and detection of any associated AMR would help to accelerate directed therapy and reduce the risk of the emergence of multidrug-resistant isolates. In this study, we identified three target genes (yhaI, epsL, and xcpW) common to K. pneumoniae isolates from both China and Europe and designed loop-mediated isothermal amplification (LAMP) assays for the detection of K. pneumoniae in clinical samples. We also designed LAMP assays for the detection of five AMR genes commonly associated with K. pneumoniae. The LAMP assays were validated on a total of 319 type reference strains and clinical isolates of diverse genetic backgrounds, in addition to 40 clinical human sputum samples, and were shown to be reliable, highly specific, and sensitive. For the K. pneumoniae-specific LAMP assay, the calculated sensitivity, specificity, and positive and negative predictive values (comparison with culture and matrix-assisted laser desorption\/ionization-time of flight mass spectrometry) were all 100% on clinical isolates and, respectively, of 100%, 91%, and 90%, and 100% when tested on clinical sputum samples, while being significantly faster than the reference methods. For the bla KPC and other carbapenemases' LAMP assays, the concordance between the LAMP results and the references methods (susceptibility tests) was 100%, on both pure cultures (n = 125) and clinical samples (n = 18). In conclusion, we developed highly sensitive and specific LAMP assays for the clinical identification of K. pneumoniae and detection of carbapenem resistance.","lamp_id":[{"id":"LPB00710","pathogen":"Klebsiella pneumoniae","target":"yhaI"},{"id":"LPB00711","pathogen":"Klebsiella pneumoniae","target":"bla KPC"}]},{"id":407,"pmid":35544541,"title":"Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application","year":2022,"journal":"PLoS One","authors":"Suying Lu, David Duplat, Paula Benitez-Bolivar, Cielo Le\u00f3n, Stephany D Villota, Eliana Veloz-Villavicencio, Valentina Ar\u00e9valo, Katariina Jaenes, Yuxiu Guo, Seray Cicek, Lucas Robinson, Philippos Peidis, Joel D Pearson, Jim Woodgett, Tony Mazzulli, Patricio Ponce, Silvia Restrepo, John M Gonz\u00e1lez, Adriana Bernal, Marcela Guevara-Suarez, Keith Pardee, Varsovia E Cevallos, Camila Gonz\u00e1lez, Rod Bremner","doi":"10.1371\/journal.pone.0268340","country":"","institute":"","deparment":"","abstract":"Continued waves, new variants, and limited vaccine deployment mean that SARS-CoV-2 tests remain vital to constrain the coronavirus disease 2019 (COVID-19) pandemic. Affordable, point-of-care (PoC) tests allow rapid screening in non-medical settings. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an appealing approach. A crucial step is to optimize testing in low\/medium resource settings. Here, we optimized RT-LAMP for SARS-CoV-2 and human \u03b2-actin, and tested clinical samples in multiple countries. \"TTTT\" linker primers did not improve performance, and while guanidine hydrochloride, betaine and\/or Igepal-CA-630 enhanced detection of synthetic RNA, only the latter two improved direct assays on nasopharygeal samples. With extracted clinical RNA, a 20 min RT-LAMP assay was essentially as sensitive as RT-PCR. With raw Canadian nasopharygeal samples, sensitivity was 100% (95% CI: 67.6% - 100%) for those with RT-qPCR Ct values \u2264 25, and 80% (95% CI: 58.4% - 91.9%) for those with 25 < Ct \u2264 27.2. Highly infectious, high titer cases were also detected in Colombian and Ecuadorian labs. We further demonstrate the utility of replacing thermocyclers with a portable PoC device (FluoroPLUM). These combined PoC molecular and hardware tools may help to limit community transmission of SARS-CoV-2.","lamp_id":[{"id":"LPB00712","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00713","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"}]},{"id":408,"pmid":32880033,"title":"Detection of Mycobacterium kansasii using a combination of loop-mediated isothermal amplification (LAMP) and lateral flow biosensors","year":2020,"journal":"International Microbiology","authors":"Chuang Chen, Jia Lu, Bo Long, Zhengyuan Rao, Yuan Gao, Weina Wang, Wenfeng Gao, Jun Yang, Shu Zhang","doi":"10.1007\/s10123-020-00143-z","country":"","institute":"","deparment":"","abstract":"Mycobacterium kansasii is an opportunistic pathogen that causes both intrapulmonary and extrapulmonary infections. The symptoms of the pulmonary diseases caused by M. kansasii closely resemble Mycobacterium tuberculosis. Rapid and accurate differentiation of M. kansasii from M. tuberculosis, as well as other mycobacteria, is crucial for developing effective therapeutics and disease treatment. In this study, we combined loop-mediated isothermal amplification (LAMP) with lateral flow biosensors (LFB) to detect M. kansasii, by targeting the species-specific sequence of rpoB, a gene which encodes the \u03b2 subunit of bacterial RNA polymerase. The assay was validated to ensure that it was highly selective by testing M. kansasii, M. tuberculosis, other species of respiratory bacteria, and other nontuberculous mycobacteria. The detection limit of the assay was 1 fg\/\u03bcL of DNA and 50 CFU of bacilli in sputum. The M. kansasii-LAMP-LFB assay is a fast, cheap, and accurate method for detecting M. kansasii by constant temperature amplification and simple interpretation.","lamp_id":[{"id":"LPB00715","pathogen":"Mycobacterium kansasii ATCC 12478","target":"rpoB"}]},{"id":409,"pmid":28514671,"title":"Evaluation of improved IS6110 LAMP assay for diagnosis of pulmonary and extra pulmonary tuberculosis","year":2017,"journal":"Journal of Microbiological Methods","authors":"Deepali Joon, Manoj Nimesh, Mandira Varma-Basil, Daman Saluja","doi":"10.1016\/j.mimet.2017.05.007","country":"","institute":"","deparment":"","abstract":"In the present study, IS6110 loop mediated isothermal amplification (LAMP) assay was modified using dUTP-UNG (uracil-DNA N-glycosylase) strategy to prevent carryover contamination, and was evaluated using clinical specimens. The clinical specimens were collected from 236 suspected patients of pulmonary tuberculosis and 315 specimens of suspected patients of extra pulmonary tuberculosis. DNA was extracted from specimens and used as template for nucleic acid amplification. The results were evaluated with culture method as gold standard. Modified IS6110 LAMP assay showed high sensitivity (94.4%) and specificity (97.2%) in specimens collected from suspected pulmonary tuberculosis patients. Sensitivity was comparatively less (86.67%) in extra pulmonary specimens while specificity was 94.04%. In conclusion, IS6110 LAMP assay was modified to prevent carry over contamination and it was validated to be rapid, sensitive and specific method with prospective application in resource-limited settings.","lamp_id":[{"id":"LPB00716","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"}]},{"id":410,"pmid":31882619,"title":"Molecular serotype-specific identification of Streptococcus pneumoniae using loop-mediated isothermal amplification","year":2019,"journal":"Scientific Reports","authors":"Chika Takano, Yoko Kuramochi, Mitsuko Seki, Dong Wook Kim, Daisuke Omagari, Mari Sasano, Bin Chang, Makoto Ohnishi, Eun Jin Kim, Kazumasa Fuwa, Paul E Kilgore, Tomonori Hoshino, Satoshi Hayakawa","doi":"10.1038\/s41598-019-56225-0","country":"","institute":"","deparment":"","abstract":"In children, the incidence of pneumococcal meningitis has decreased since the introduction of pneumococcal conjugate vaccine (PCV7 and PCV13). However, since the introduction of the vaccine, developed countries have seen the emergence of non-PCV13 serotypes. However, invasive pneumococcal disease (IPD) caused by PCV13-targeted serotypes still represents an important public health problem in resource-limited countries. To develop a rapid, simple, and cost-effective assay to detect serotypes of Streptococcus pneumoniae, we developed a novel loop-mediated isothermal amplification (LAMP) assay based on the sequences available for the 13 capsular types that are included in PCV13: 1, 3, 4, 5, 6 A, 6B, 7 F, 9 V, 14, 18 C, 19 A, 19 F, and 23 F. We evaluated test reactivity, specificity, sensitivity and performance, and compared the results between established LAMP and conventional PCR assays. To support its clinical use, the detection limits of the LAMP assay were evaluated using bacterial genomic DNA-spiked cerebrospinal fluid (CSF) and blood specimens. We confirmed the specificity of the LAMP assay using 41 serotypes of pneumococcal strains. The sensitivity of the LAMP assay was 10 to 100 copies per reaction, compared to 10 to 10^4 copies per reaction for PCR assays. The detection limits of the LAMP assay were comparable when using DNA-spiked CSF and blood specimens, as compared to using purified DNA as the template. In conclusion, a rapid and simple LAMP-based pneumococcal serotyping method has been developed. This is the first report of a LAMP method for a PCV13 serotype-specific identification assay, which could be a promising step to facilitate epidemiological studies of pneumococcal serotyping.","lamp_id":[{"id":"LPB00717","pathogen":"Streptococcus pneumoniae","target":"wzy"},{"id":"LPB00718","pathogen":"Streptococcus pneumoniae","target":"wciP"},{"id":"LPB00719","pathogen":"Streptococcus pneumoniae","target":"wzy"},{"id":"LPB00720","pathogen":"Streptococcus pneumoniae","target":"wzy"},{"id":"LPB00721","pathogen":"Streptococcus pneumoniae","target":"wzy"},{"id":"LPB00722","pathogen":"Streptococcus pneumoniae","target":"wzy"},{"id":"LPB00723","pathogen":"Streptococcus pneumoniae","target":"wzy"},{"id":"LPB00724","pathogen":"Streptococcus pneumoniae","target":"wzy"},{"id":"LPB00725","pathogen":"Streptococcus pneumoniae","target":"galU"},{"id":"LPB00726","pathogen":"Streptococcus pneumoniae","target":"wzy"},{"id":"LPB00727","pathogen":"Streptococcus pneumoniae","target":"wzy"},{"id":"LPB00728","pathogen":"Streptococcus pneumoniae","target":"wzy"}]},{"id":411,"pmid":31653930,"title":"A modular paper-and-plastic device for tuberculosis nucleic acid amplification testing in limited-resource settings","year":2019,"journal":"Scientific Reports","authors":"Navjot Kaur, Joy S Michael, Bhushan J Toley","doi":"10.1038\/s41598-019-51873-8","country":"","institute":"","deparment":"","abstract":"We present a prototype for conducting rapid, inexpensive and point-of-care-compatible nucleic acid amplification tests (NAATs) for tuberculosis (TB). The fluorescent isothermal paper-and-plastic NAAT (FLIPP-NAAT) uses paper-based loop mediated isothermal amplification (LAMP) for DNA detection. The cost of materials required to build a 12-test-zone device is $0.88 and the cost of reagents per reaction is $0.43. An inexpensive imaging platform enables filter-free fluorescence detection of amplified DNA using a cell-phone camera. FLIPP-NAAT can be operated by an untrained user and only requires a regular laboratory incubator as ancillary equipment. All reagents can be dry-stored in the device, facilitating storage and transportation without cold chains. The device design is modular and the assay demonstrated high specificity to Mycobacterium tuberculosis (Mtb), analytical sensitivity of the order of 10 copies of Mtb gDNA, and tolerance to complex samples. The clinical sensitivity and specificity of sputum-based FLIPP NAAT tests were 100% (zero false negatives) and 68.75% (five false positives), respectively (N = 30), using Xpert MTB\/RIF assay as the reference standard. FLIPP-NAAT has the potential to provide affordable and accessible molecular diagnostics for TB in low- and middle-income countries, when used in conjunction with an appropriate sample preparation technique. Although demonstrated for the detection of TB, FLIPP-NAAT is a platform technology for amplification of any nucleic acid sequence.","lamp_id":[{"id":"LPB00729","pathogen":"Mycobacterium tuberculosis H37Rv","target":"hspX"}]},{"id":412,"pmid":22395179,"title":"A portable and integrated nucleic acid amplification microfluidic chip for identifying bacteria","year":2012,"journal":"Lab on a Chip","authors":"Xueen Fang, Hui Chen, Lingjia Xu, Xingyu Jiang, Wenjuan Wu, Jilie Kong","doi":"10.1039\/c2lc40055c","country":"","institute":"","deparment":"","abstract":"In this work, we developed a portable integrated microchip of loop-mediated isothermal nucleic acid amplification (LAMP). This chip, with sample-to-answer capability, could perform rapid DNA release, exponential signal amplification and naked-eye result read-out in single or multiplex format. We call it i\u03bcLAMP, namely integrated micro-LAMP, which was successfully used for point-of-care identification of bacteria.","lamp_id":[{"id":"LPB00730","pathogen":"Mycobacterium tuberculosis H37Rv","target":"16S rRNA"}]},{"id":413,"pmid":27660018,"title":"Sample-to-answer on molecular diagnosis of bacterial infection using integrated lab-on-a-disc","year":2017,"journal":"Biosensors and Bioelectronics","authors":"J F C Loo, H C Kwok, C C H Leung, S Y Wu, I L G Law, Y K Cheung, Y Y Cheung, M L Chin, P Kwan, M Hui, S K Kong, H P Ho","doi":"10.1016\/j.bios.2016.09.001","country":"","institute":"","deparment":"","abstract":"Sepsis by bacterial infection causes high mortality in patients in intensive care unit (ICU). Rapid identification of bacterial infection is essential to ensure early appropriate administration of antibiotics to save lives of patients, yet the present benchtop molecular diagnosis is time-consuming and labor-intensive, which limits the treatment efficiency especially when the number of samples to be tested is extensive. Therefore, we hereby report a microfluidic platform lab-on-a-disc (LOAD) to provide a sample-to-answer solution. Our LOAD customized design of microfluidic channels allows automation to mimic sequential analytical steps in benchtop environment. It relies on a simple but controllable centrifugation force for the actuation of samples and reagents. Our LOAD system performs three major functions, namely DNA extraction, isothermal DNA amplification and real-time signal detection, in a predefined sequence. The disc is self-contained for conducting sample heating with chemical lysis buffer and silica microbeads are employed for DNA extraction from clinical specimens. Molecular diagnosis of specific target bacteria DNA sequences is then performed using a real-time loop-mediated isothermal amplification (RT-LAMP) with SYTO-9 as the signal reporter. Our LOAD system capable of bacterial identification of Mycobacterium tuberculosis (TB) and Acinetobacter baumanii (Ab) with the detection limits 10^3 cfu\/mL TB in sputum and 10^2 cfu\/mL Ab in blood within 2h after sample loading. The reported LOAD based on an integrated approach should address the growing needs for rapid point-of-care medical diagnosis in ICU.","lamp_id":[{"id":"LPB00731","pathogen":"Mycobacterium tuberculosis","target":"IS6110"},{"id":"LPB00732","pathogen":"Acinetobacter baumannii","target":null}]},{"id":414,"pmid":25533219,"title":"Real-time fluorescence Loop-Mediated Isothermal Amplification (LAMP) for rapid and reliable diagnosis of pulmonary tuberculosis","year":2015,"journal":"Journal of Microbiological Methods","authors":"Donglin Cao, Liangshan Hu, Maorui Lin, Mingyou Li, Zebing Ye, Hongtao Sun, Jiwei Huang, Huawen Yang, Junzhang Tian","doi":"10.1016\/j.mimet.2014.12.013","country":"","institute":"","deparment":"","abstract":"A reliable, simple and rapid diagnostic method that can be helpful in pulmonary tuberculosis diagnosis is urgently needed. Loop-mediated Isothermal Amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. In this study, real-time fluorescence LAMP was evaluated to rapidly detect Mycobacterium tuberculosis in sputum and was compared to the performance of real-time fluorescence quantitative PCR (Q-PCR). All the standard MTB strains were successfully detected and limit of detection (LOD) was 10(2)CFU\/mL by real-time fluorescence LAMP within 20min. In light of MTB in sputum, the real-time fluorescence LAMP method yielded a sensitivity of 98.0% and a specificity of 78.3%, compared to Q-PCR assay, which yielded a sensitivity of 96.0% and a specificity of 82.6% for PTB diagnosis. There was an excellent overall agreement between LAMP and Q-PCR for PTB (\u03ba=0.315) and non-PTB (\u03ba=0.862). Therefore, the real-time fluorescence LAMP assay is a rapid, sensitive, and specific method to detect pulmonary tuberculosis.","lamp_id":[{"id":"LPB00734","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"}]},{"id":415,"pmid":33591996,"title":"23-valent polysaccharide vaccine (PPSV23)-targeted serotype-specific identification of Streptococcus pneumoniae using the loop-mediated isothermal amplification (LAMP) method","year":2021,"journal":"PLoS One","authors":"Jiwon Lee, Youngbae Yoon, Eun Jin Kim, Donghyun Lee, Yeongjun Baek, Chika Takano, Bin Chang, Takahiro Iijima, Paul E Kilgore, Satoshi Hayakawa, Tomonori Hoshino, Dong Wook Kim, Mitsuko Seki","doi":"10.1371\/journal.pone.0246699","country":"","institute":"","deparment":"","abstract":"Reports of invasive disease due to Streptococcus pneumoniae have declined since the introduction of pneumococcal conjugate vaccines (PCV7 and PCV13). The incidence of invasive diseases due to S. pneumoniae that are not addressed by the vaccines, however, has increased in children and adults, creating a global public health problem. Previously, we established the loop-mediated isothermal amplification (LAMP) method for a PCV13 serotype-specific assay. In the current study, we developed a rapid, simple, and cost-effective assay to detect serotypes in the 23-valent pneumococcal polysaccharide vaccine (PPSV23) using the LAMP method. In this study, LAMP primer sets for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F of S. pneumoniae were developed. The reactivity, specificity, and sensitivity of LAMP assays were determined and compared to those of conventional PCR. The feasibility of LAMP assays in clinical application in patients with invasive pneumococcal diseases was validated by defining the detection limit of the LAMP assay with bacterial genomic DNA-spiked blood specimens. The specificity of each LAMP assay was determined using 44 serotypes of pneumococcal strains. Their sensitivity was 100 copies per reaction versus 10^3 to 10^6 copies per reaction for PCR assays. Using DNA-spiked blood specimens, excluding the LAMP assay that targeted serotype 22F (10^3 copies per reaction), the limit of detection of the LAMP assay was similar to that with purified DNA as the template (10^2 copies per reaction), compared with 10^3 to >10^6 copies per reaction for PCR assays. In conclusion, a rapid and simple LAMP-based PPSV23-targeted serotype detection assay was developed for use in many countries. This study is the first report of a LAMP-based assay for identification of PPSV23 serotypes. Further evaluation of this assay is needed through surveillance and vaccine efficacy studies.","lamp_id":[{"id":"LPB00735","pathogen":"Streptococcus pneumoniae","target":"wzy"},{"id":"LPB00736","pathogen":"Streptococcus pneumoniae","target":"wzy"},{"id":"LPB00737","pathogen":"Streptococcus pneumoniae","target":"wzx"},{"id":"LPB00738","pathogen":"Streptococcus pneumoniae","target":"wcrG"},{"id":"LPB00739","pathogen":"Streptococcus pneumoniae","target":"wzy"},{"id":"LPB00740","pathogen":"Streptococcus pneumoniae","target":"mnaB"},{"id":"LPB00741","pathogen":"Streptococcus pneumoniae","target":"wzy"},{"id":"LPB00742","pathogen":"Streptococcus pneumoniae","target":"wciP"},{"id":"LPB00743","pathogen":"Streptococcus pneumoniae","target":"wciL"},{"id":"LPB00744","pathogen":"Streptococcus pneumoniae","target":"wcwV"},{"id":"LPB00745","pathogen":"Streptococcus pneumoniae","target":"wzy"}]},{"id":416,"pmid":34935909,"title":"A Loop-mediated Isothermal Amplification With a Nanoparticle-Based Lateral Flow Biosensor Assay to Detect Pseudomonas aeruginosa in Endophthalmitis","year":2021,"journal":"Translational Vision Science & Technology","authors":"Kui Dong, ZhiMing Kang, Xuan Ji, Xinxin Zhang, PeiNi Cheng, Bin Sun","doi":"10.1167\/tvst.10.14.26","country":"","institute":"","deparment":"","abstract":"Purpose: Pseudomonas aeruginosa is the most common bacteria causing endophthalmitis after cataract surgery. Vitreous fluid culture and molecular studies are commonly used in clinical diagnoses, but have disadvantages, such as a long culture cycle and low detection sensitivity. Here, we report a loop-mediated isothermal amplification (LAMP) method combined with the nanoparticles-lateral flow biosensor (LFB) method for rapid and specific detection of P. aeruginosa.\n\nMethods: A set of six primers was designed to target the OprL gene of P. aeruginosa. Genomic DNA extracted from several gram-negative and gram-positive bacteria was used to determine the sensitivity and specificity of the analysis. LAMP reactions were conducted at 65 \u00b0C for 50 minutes, and results were reported using the LFB method.\n\nResults: The DNA template of P. aeruginosa was specifically recognized by the P. aeruginosa-LAMP-LFB (PA-LAMP-LFB) method as no cross reactions were observed for non-P. aeruginosa templates. The analytical sensitivity of our assay was 100 fg per test for the pure cultured DNA template, and the result obtained using the LFB was consistent with that of colorimetric indicator detection. The whole test could be completed within 1h. This method was used to detect P. aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae; only P. aeruginosa was positive. The positive rates of P. aeruginosa detected by a traditional culture method, the LAMP-LFB method, and the fluorescence quantitative polymerase chain reaction method were 17.7%, 17.7%, and 13.3%, respectively.\n\nConclusions: The P. aeruginosa-LAMP-LFB method established here is a rapid, specific, and sensitive method for the detection of P. aeruginosa, which can be widely used.","lamp_id":[{"id":"LPB00746","pathogen":"Pseudomonas aeruginosa PAO1","target":"OprL"}]},{"id":417,"pmid":35250283,"title":"A New Method Based on LAMP-CRISPR-Cas12a-Lateral Flow Immunochromatographic Strip for Detection","year":2022,"journal":"Infection and Drug Resistance","authors":"Huaming Xu, Hao Tang, Rongrong Li, Zhaoxin Xia, Wensu Yang, Yi Zhu, Zhen Liu, Guoping Lu, Shenwang Ni, Jilu Shen","doi":"10.2147\/IDR.S348456","country":"","institute":"","deparment":"","abstract":"Introduction: Carbapenemase-mediated antimicrobial resistance is currently a hot spot of global concern. Carbapenem-resistant organisms are highly prevalent in hospitals associated with difficult-to-treat infections, resulting in poor clinical outcome due to limited treatment options. It is urgently needed to have a rapid, efficient, and convenient molecular assay for identifying such resistant strains.\n\nMethods: For this end, we developed a new laboratory assay targeting Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-\u03b2-lactamase (NDM) based on loop-mediated isothermal amplification, CRISPR-Cas12a, and lateral flow immunochromatographic strip (CRISPR-Cas-LAMP-lateral flow strip). The method was designed to use a guide RNA (gRNA) to recognize the target DNA and guide Cas12a to cleave the target DNA, and simultaneously cleave any single-stranded DNA within the cleavage reaction system.\n\nResults: The cleavage products are visible to the naked eye on the lateral flow strip. This method is highly sensitive in direct detection of bacteria in samples containing at least 3\u00d7105 CFU\/mL without the need for bacterial culture.\n\nDiscussion: It provides shorter turnaround time and higher specificity than the conventional bacterial culture and susceptibility testing method. This new assay is applicable for extensive use in hospital infection control, as well as identification and treatment of resistant strains due to simple operation and inexpensive apparatuses.","lamp_id":[{"id":"LPB00747","pathogen":"Klebsiella pneumoniae","target":"KPC"},{"id":"LPB00748","pathogen":"Klebsiella pneumoniae","target":"NDM"}]},{"id":418,"pmid":21847513,"title":"LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila","year":2011,"journal":"Applied Microbiology and Biotechnology","authors":"Xi Lu, Zi-Yao Mo, Hong-Bo Zhao, He Yan, Lei Shi","doi":"10.1007\/s00253-011-3496-8","country":"","institute":"","deparment":"","abstract":"Legionella pneumophila is accounted for more than 80% of Legionella infection. However it is difficult to discriminate between the L. pneumophila and non-L. pneumophila species rapidly. In order to detect the Legionella spp. and distinguish L. pneumophila from Legionella spp., a real-time loop-mediated isothermal amplification (LAMP) platform that targets a specific sequence of the 16S rRNA gene was developed. LS-LAMP amplifies the fragment of the 16S rRNA gene to detect all species of Legionella genus. A specific sequence appears at the 16S rRNA gene of L. pneumophila, while non-L. pneumophila strains have a variable sequence in this site, which can be recognized by the primer of LP-LAMP. In the present study, 61 reference strains were used for the method verification. We found that the specificity was 100% for both LS-LAMP and LP-LAMP, and the sensitivity of LAMP assay for L. pneumophila detection was between 52 and 5.2 copies per reaction. In the environmental water samples detection, a total of 107 water samples were identified by the method. The culture and serological test were used as reference methods. The specificity of LS-LAMP and LP-LAMP for the samples detection were 91.59% (98\/107) and 93.33% (56\/60), respectively. The sensitivity of LS-LAMP and LP-LAMP were 100% (51\/51) and 100% (18\/18). The results suggest that real-time LAMP, as a new assay, provides a specific and sensitive method for rapid detection and differentiation of Legionella spp. and L. pneumophila and should be utilized to test environmental water samples for increased rates of detection.","lamp_id":[{"id":"LPB00749","pathogen":"Legionella pneumophila","target":"16S rRNA"}]},{"id":419,"pmid":31940771,"title":"Assessment of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Pathogenic Bacteria from Respiratory Samples in Patients with Hospital-Acquired Pneumonia","year":2020,"journal":"Microorganisms","authors":"Andrea Vergara, Herv\u00e9 Boutal, Adri\u00e1n Ceccato, M\u00edriam L\u00f3pez, Adri\u00e0 Cruells, Leticia Bueno-Freire, Javier Moreno-Morales, Jorge Puig de la Bellacasa, Pedro Castro, Antoni Torres, Francesc Marco, Climent Casals-Pascual, Jordi Vila","doi":"10.3390\/microorganisms8010103","country":"","institute":"","deparment":"","abstract":"Rapid identification of the causative agent of hospital-acquired pneumonia (HAP) will allow an earlier administration of a more appropriate antibiotic and could improve the outcome of these patients. The aim of this study was to develop a rapid protocol to identify the main microorganisms involved in HAP by loop-mediated isothermal amplification (LAMP) directly from respiratory samples. First of all, a rapid procedure (<30 min) to extract the DNA from bronchoalveolar lavage (BAL), endotracheal aspirate (EA) or bronchoaspirate (BAS) was set up. A specific LAMP for Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii was performed with the extracted solution at 65 \u00b0C for 30-40 min. Overall, 58 positive BAL and 83 EA\/BAS samples were tested. The limits of detection varied according to the microorganism detected. Validation of the LAMP assay with BAL samples showed that the assay was 100% specific and 86.3% sensitive (positive predictive value of 100% and a negative predictive value of 50%) compared with culture. Meanwhile for BAS\/EA samples, the assay rendered the following statistical parameters: 100% specificity, 94.6% sensitivity, 100% positive predictive value and 69.2% negative predictive value. The turnaround time including sample preparation and LAMP was circa 1 h. LAMP method may be used to detect the most frequent bacteria causing HAP. It is a simple, cheap, sensitive, specific and rapid assay.","lamp_id":[{"id":"LPB00750","pathogen":"Klebsiella pneumoniae subsp. pneumoniae MGH 78578","target":"KPN_04473"},{"id":"LPB00751","pathogen":"Pseudomonas aeruginosa","target":"oprI"},{"id":"LPB00752","pathogen":"Acinetobacter baumannii ATCC 17978","target":"AdeS"},{"id":"LPB00753","pathogen":"Stenotrophomonas maltophilia","target":"StmPr2"}]},{"id":420,"pmid":22416993,"title":"A loop-mediated isothermal amplification method for rapid detection of NDM-1 gene","year":2012,"journal":"Microbial Drug Resistance","authors":"Jing Qi, Yijun Du, Xiaoling Zhu, Hua Bai, Yanbo Luo, Yuqing Liu","doi":"10.1089\/mdr.2011.0220","country":"","institute":"","deparment":"","abstract":"A loop-mediated isothermal amplification (LAMP) method was developed for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-\u03b2-lactamase-1 (NDM-1), with its specificity and sensitivity having been evaluated. Six primers, including a pair of outer primers, a pair of inner primers, and a pair of loop primers, were specially designed for recognizing eight distinct sequences on the target NDM-1 gene. The amplification reaction was performed within only 40 min under isothermal conditions at 65\u00b0C in a regular water bath. The LAMP assay showed good specificity and higher sensitivity than the conventional polymerase chain reaction (PCR), with a detection limit of 1 pg genomic DNA per tube of one NDM-1-positive reference strain. The detection result for the 345 clinical samples showed 100% consistence with the result by the PCR method, and three contaminated samples could be detected correctly by LAMP assays, while they could not be detected by PCR. The LAMP method reported here demonstrated a potential and valuable means for detection of the NDM-1 gene: easy, rapid, visual, specific, accurate, and sensitive, especially useful for on-the-spot investigation.","lamp_id":[{"id":"LPB00755","pathogen":"Klebsiella pneumoniae","target":"NDM"}]},{"id":421,"pmid":24789191,"title":"Development and Clinical Evaluation of sdaA Loop-Mediated Isothermal Amplification Assay for Detection of Mycobacterium tuberculosis with an Approach To Prevent Carryover Contamination","year":2014,"journal":"Journal of Clinical Microbiology","authors":"Manoj Nimesh, Deepali Joon, Mandira Varma-Basil, Daman Saluja","doi":"10.1128\/JCM.00907-14","country":"","institute":"","deparment":"","abstract":"A rapid and sensitive loop-mediated isothermal amplification assay for the sdaA gene of Mycobacterium tuberculosis was developed using a dUTP-uracil-N-glycosylase (dUTP-UNG) strategy to prevent carryover contamination. Evaluation of the assay using clinical specimens (n = 648) showed high specificity (97.2%) and sensitivity (100%), demonstrating its potential as a diagnostic test for tuberculosis, especially in resource-limited settings.","lamp_id":[{"id":"LPB00756","pathogen":"Mycobacterium tuberculosis H37Rv","target":"sdaA"}]},{"id":422,"pmid":30858005,"title":"Development and evaluation of rapid and specific sdaA LAMP-LFD assay with Xpert MTB\/RIF assay for diagnosis of tuberculosis","year":2019,"journal":"Journal of Microbiological Methods","authors":"Deepali Joon, Manoj Nimesh, Shraddha Gupta, Chanchal Kumar, Mandira Varma-Basil, Daman Saluja","doi":"10.1016\/j.mimet.2019.03.002","country":"","institute":"","deparment":"","abstract":"There is need for rapid and cost-effective diagnostic test for tuberculosis. The present study was carried out to design a Loop-mediated isothermal amplification (LAMP) assay combined with lateral flow dipstick (LFD) as a point-of-care method for diagnosis of TB. LAMP assay targeting sdaA gene combined with LFD for sequence specific detection was standardized in user friendly and rapid format. It does not require sophisticated instruments and shows visual results instantly. The LAMP-LFD assay was validated using culture confirmed specimens. The assay was evaluated in a cross-sectional study using respiratory specimens collected from patients in Delhi, India and it showed high concordance with GeneXpert MTB\/RIF assay. Lateral flow dipstick method has provided an excellent detection format with LAMP method. The LAMP-LFD assay showed high diagnostic accuracy in comparison to other methods and can be used as a point-of-care test in cost-effective manner.","lamp_id":[{"id":"LPB00757","pathogen":"Mycobacterium tuberculosis H37Rv","target":"sdaA"}]},{"id":423,"pmid":31449526,"title":"Loop-mediated Isothermal Amplification and nested PCR of the Internal Transcribed Spacer (ITS) for Histoplasma capsulatum detection","year":2019,"journal":"PLOS Neglected Tropical Diseases","authors":"Matheus da Silva Zatti, Thales Domingos Arantes, Jos\u00e9 Alex Louren\u00e7o Fernandes, M\u00f4nica Baumgardt Bay, Eveline Pipolo Milan, Georggia Fatima Silva Naliato, Raquel Cordeiro Theodoro","doi":"10.1371\/journal.pntd.0007692","country":"","institute":"","deparment":"","abstract":"Background: Histoplasmosis is a neglected disease that affects mainly immunocompromised patients, presenting a progressive dissemination pattern and a high mortality rate, mainly due to delayed diagnosis, caused by slow fungal growth in culture. Therefore, a fast, suitable and cost-effective assay is required for the diagnosis of histoplasmosis in resource-limited laboratories. This study aimed to develop and evaluate two new molecular approaches for a more cost-effective diagnosis of histoplasmosis.\n\nMethodology: Seeking a fast, suitable, sensitive, specific and low-cost molecular detection technique, we developed a new Loop-mediated Isothermal Amplification (LAMP) assay and nested PCR, both targeting the Internal Transcribed Spacer (ITS) multicopy region of Histoplasma capsulatum. The sensitivity was evaluated using 26 bone marrow and 1 whole blood specimens from patients suspected to have histoplasmosis and 5 whole blood samples from healthy subjects. All specimens were evaluated in culture, as a reference standard test, and Hcp100 nPCR, as a molecular reference test. A heparin-containing whole blood sample from a heathy subject was spiked with H. capsulatum cells and directly assayed with no previous DNA extraction.\n\nResults: Both assays were able to detect down to 1 fg\/\u03bcL of H. capsulatum DNA, and ITS LAMP results could also be revealed to the naked-eye by adding SYBR green to the reaction tube. In addition, both assays were able to detect all clades of Histoplasma capsulatum cryptic species complex. No cross-reaction with other fungal pathogens was presented. In comparison with Hcp100 nPCR, both assays reached 83% sensitivity and 92% specificity. Furthermore, ITS LAMP assay showed no need for DNA extraction, since it could be directly applied to crude whole blood specimens, with a limit of detection of 10 yeasts\/\u03bcL.\n\nConclusion: ITS LAMP and nPCR assays have the potential to be used in conjunction with culture for early diagnosis of progressive disseminated histoplasmosis, allowing earlier, appropriate treatment of the patient. The possibility of applying ITS LAMP, as a direct assay, with no DNA extraction and purification steps, makes it suitable for resource-limited laboratories. However, more studies are necessary to validate ITS LAMP and nPCR as direct assay in other types of clinical specimens.","lamp_id":[{"id":"LPB00758","pathogen":"Histoplasma capsulatum","target":"ITS1"}]},{"id":424,"pmid":25708458,"title":"Robust strand exchange reactions for the sequence-specific, real-time detection of nucleic acid amplicons","year":2015,"journal":"Analytical Chemistry","authors":"Yu Sherry Jiang, Sanchita Bhadra, Bingling Li, Yuefeng Rose Wu, John N Milligan, Andrew D Ellington","doi":"10.1021\/ac504387c","country":"","institute":"","deparment":"","abstract":"Loop-mediated isothermal amplification (LAMP) of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 10(9) copies from less than 100 copies of template DNA within an hour. Unfortunately, although the amplification reactions are extremely powerful, real-time and specific detection of LAMP products remains analytically challenging. In order to both improve the specificity of LAMP detection and to make readout simpler and more reliable, we have replaced the intercalating dye typically used for monitoring in real-time fluorescence with a toehold-mediated strand exchange reaction termed one-step strand displacement (OSD). Due to the inherent sequence specificity of toehold-mediated strand exchange, the OSD reporter could successfully distinguish side products from true amplicons arising from templates corresponding to the biomedically relevant M. tuberculosis RNA polymerase (rpoB) and the melanoma-related biomarker BRAF. OSD allowed the Yes\/No detection of rpoB in a complex mixture such as synthetic sputum and also demonstrated single nucleotide specificity in Yes\/No detection of a mutant BRAF allele (V600E) in the presence of 20-fold more of the wild-type gene. Real-time detection of different genes in multiplex LAMP reactions also proved possible. The development of simple, readily designed, modular equivalents of TaqMan probes for isothermal amplification reactions should generally improve the applicability of these reactions and may eventually assist with the development of point-of-care tests.","lamp_id":[{"id":"LPB00759","pathogen":"Mycobacterium tuberculosis H37Rv","target":"rpoB"}]},{"id":425,"pmid":24478477,"title":"Development of a loop-mediated isothermal amplification method for detection of Histoplasma capsulatum DNA in clinical samples","year":2014,"journal":"Journal of Clinical Microbiology","authors":"Christina M Scheel, Yitian Zhou, Raquel C Theodoro, Bethany Abrams, S Arunmozhi Balajee, Anastasia P Litvintseva","doi":"10.1128\/JCM.02739-13","country":"","institute":"","deparment":"","abstract":"Improved methods for the detection of Histoplasma capsulatum are needed in regions with limited resources in which the organism is endemic, where delayed diagnosis of progressive disseminated histoplasmosis (PDH) results in high mortality rates. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid inexpensive molecular diagnosis of this disease. Primers for LAMP were designed to amplify the Hcp100 locus of H. capsulatum. The sensitivity and limit of detection were evaluated using DNA extracted from 91 clinical isolates of known geographic subspecies, while the assay specificity was determined using DNA extracted from 50 other fungi and Mycobacterium tuberculosis. Urine specimens (n = 6) collected from HIV-positive individuals with culture- and antigen-proven histoplasmosis were evaluated using the LAMP assay. Specimens from healthy persons (n = 10) without evidence of histoplasmosis were used as assay controls. The Hcp100 LAMP assay was 100% sensitive and specific when tested with DNA extracted from culture isolates. The median limit of detection was \u22646 genomes (range, 1 to 300 genomes) for all except one geographic subspecies. The LAMP assay detected Hcp100 in 67% of antigen-positive urine specimens (4\/6 specimens), and results were negative for Hcp100 in all healthy control urine specimens. We have shown that the Hcp100 LAMP assay is a rapid affordable assay that can be used to expedite culture confirmation of H. capsulatum in regions in which PDH is endemic. Further, our results indicate proof of the concept that the assay can be used to detect Histoplasma DNA in urine. Further evaluation of this assay using body fluid samples from a larger patient population is warranted.","lamp_id":[{"id":"LPB00760","pathogen":"Histoplasma capsulatum","target":"Hcp100"}]},{"id":426,"pmid":29436623,"title":"Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa","year":2018,"journal":"Molecular Medicine Reports","authors":"Orapan Manajit, Siwaporn Longyant, Paisarn Sithigorngul, Parin Chaivisuthangkura","doi":"10.3892\/mmr.2018.8557","country":"","institute":"","deparment":"","abstract":"Pseudomonas aeruginosa (P. aeruginosa) is an important opportunistic pathogen that causes serious infections in humans, including keratitis in contact lens wearers. Therefore, establishing a rapid, specific and sensitive method for the identification of P. aeruginosa is imperative. In the present study, the uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification combined with nanogold labeled hybridization probe (UDG-LAMP-AuNP) was developed for the detection of P. aeruginosa. UDG-LAMP was performed to prevent carry over contamination and the LAMP reactions can be readily observed using the nanogold probe. A set of 4 primers and a hybridization probe were designed based on the ecfX gene. The UDG-LAMP reactions were performed at 65\u02daC for 60 min using the ratio of 40% deoxyuridine triphosphate to 60% deoxythymidine triphosphate. The detection of UDG-LAMP products using the nanogold labeled hybridization probe, which appeared as a red-purple color, was examined at 65\u02daC for 5 min with 40 mM MgSO4. The UDG-LAMP-AuNP demonstrated specificity to all tested isolates of P. aeruginosa without cross reaction to other bacteria. The sensitivity for the detection of pure culture was 1.6x10^3 colony-forming units (CFU) ml-1 or equivalent to 3 CFU per reaction while that of polymerase chain reaction was 30 CFU per reaction. The detection limit of spiked contact lenses was 1.1x10^3 CFU ml-1 or equivalent to 2 CFU per reaction. In conclusion, the UDG-LAMP-AuNP assay was rapid, simple, specific and was effective for the identification of P. aeruginosa in contaminated samples.","lamp_id":[{"id":"LPB00761","pathogen":"Pseudomonas aeruginosa","target":"ecfX"}]},{"id":427,"pmid":26522754,"title":"Rapid detection of Pseudomonas aeruginosa and Acinetobacter baumannii Harboring bla(VIM-2), bla(IMP-1) and bla(OXA-23) genes by using loop-mediated isothermal amplification methods","year":2016,"journal":"Annals of Laboratory Medicine","authors":"Hye Jin Kim, Hyung Sun Kim, Jae Myun Lee, Sang Sun Yoon, Dongeun Yong","doi":"10.3343\/alm.2016.36.1.15","country":"","institute":"","deparment":"","abstract":"Background: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of bla(VIM-2), bla(IMP-1), and bla(OXA-23), which are critical components for carbapenem resistance.\n\nMethods: Two sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO\u2084) by testing different concentrations of MgSO\u2084. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized bla(VIM-2), bla(IMP-1), and bla(OXA-23) LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates.\n\nResults: Only one strain of the 100 CRPA isolates harbored bla(IMP-1), whereas none of them harbored bla(VIM-2). These results indicate that the acquisition of bla(VIM-2) or bla(IMP-1) may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained bla(OXA-23), but none contained bla(VIM-2) and bla(IMP-1) alleles.\n\nConclusions: Our results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.","lamp_id":[{"id":"LPB00762","pathogen":"Pseudomonas aeruginosa","target":"blaVIM-2"},{"id":"LPB00763","pathogen":"Pseudomonas aeruginosa","target":"blaIMP-1"},{"id":"LPB00764","pathogen":"Acinetobacter baumannii","target":"blaOXA-23"}]},{"id":428,"pmid":23843955,"title":"Real-time fluorescence loop mediated isothermal amplification for the detection of Acinetobacter baumannii","year":2013,"journal":"PLoS One","authors":"Qinqin Wang, Yanbin Zhou, Shaoli Li, Chao Zhuo, Siqi Xu, Lixia Huang, Ling Yang, Kang Liao","doi":"10.1371\/journal.pone.0066406","country":"","institute":"","deparment":"","abstract":"Background: Detection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii.\n\nMethodology and significant findings: Species-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively.\n\nConclusion: The RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has great potential of field use as a molecular tool for detection of A. baumannii.","lamp_id":[{"id":"LPB00765","pathogen":"Acinetobacter baumannii","target":"pgaD"}]},{"id":429,"pmid":26441924,"title":"Rapid detection of Acinetobacter baumannii and molecular epidemiology of carbapenem-resistant A. baumannii in two comprehensive hospitals of Beijing, China","year":2015,"journal":"Frontiers in Microbiology","authors":"Puyuan Li, Wenkai Niu, Huan Li, Hong Lei, Wei Liu, Xiangna Zhao, Leijing Guo, Dayang Zou, Xin Yuan, Huiying Liu, Jing Yuan, Changqing Bai","doi":"10.3389\/fmicb.2015.00997","country":"","institute":"","deparment":"","abstract":"Acinetobacter baumannii is an important opportunistic pathogen associated with a variety of nosocomial infections. A rapid and sensitive molecular detection in clinical isolates is quite needed for the appropriate therapy and outbreak control of A. baumannii. Group 2 carbapenems have been considered the agents of choice for the treatment of multiple drug-resistant A. baumannii. But the prevalence of carbapenem-resistant A. baumannii (CRAB) has been steadily increasing in recent years. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of A. baumannii in clinical samples by using high-specificity primers of the bla OXA-51 gene. Then we investigated the OXA-carbapenemases molecular epidemiology of A. baumannii isolates in two comprehensive hospitals in Beijing. The results showed that the LAMP assay could detect target DNA within 60 min at 65\u00b0C. The detection limit was 50 pg\/\u03bcl, which was about 10-fold greater than that of PCR. Furthermore, this method could distinguish A. baumannii from the homologous A. nosocomialis and A. pittii. A total of 228 positive isolates were identified by this LAMP-based method for A. baumannii from 335 intensive care unit patients with clinically suspected multi-resistant infections in two hospitals in Beijing. The rates of CRAB are on the rise and are slowly becoming a routine phenotype for A. baumannii. Among the CRABs, 92.3% harbored both the bla OXA-23 and bla OXA-51 genes. Thirty-three pulsotypes were identified by pulsed-field gel electrophoresis, and the majority belonged to clone C. In conclusion, the LAMP method developed for detecting A. baumannii was faster and simpler than conventional PCR and has great potential for both point-of-care testing and basic research. We further demonstrated a high distribution of class D carbapenemase-encoding genes, mainly OXA-23, which presents an emerging threat in hospitals in China.","lamp_id":[{"id":"LPB00766","pathogen":"Acinetobacter baumannii","target":"blaOXA-51"}]},{"id":430,"pmid":31832709,"title":"Loop-mediated amplification as promising on-site detection approach for Legionella pneumophila and Legionella spp","year":2020,"journal":"Applied Microbiology and Biotechnology","authors":"Cornelia Reuter, Nicole Slesiona, Stefanie Hentschel, Oliver Aehlig, Antje Breitenstein, Andrea Cs\u00e1ki, Thomas Henkel, Wolfgang Fritzsche","doi":"10.1007\/s00253-019-10286-3","country":"","institute":"","deparment":"","abstract":"Recently Legionella pneumophila is the main causative waterborne organism of severe respiratory infections. Additionally, other Legionella species are documented as human pathogens. In our work, we describe a rapid detection method which combines two advantages for sensitive and specific detection of the genus Legionella: the fast isothermal amplification method \"Loop-mediated isothermal AMPlification\" (LAMP), and a colorimetric detection method using the metal indicator hydroxynaphtol blue (HBN) which allows to determine an optical signal with a simple readout (with the naked eye). Moreover, we present two approaches for minimizing the assay volume using a stationary microchip LAMP and droplet digital-based LAMP (ddLAMP) as promising highly sensitive setups.","lamp_id":[{"id":"LPB00767","pathogen":"Legionella pneumophila","target":"16S rRNA"}]},{"id":431,"pmid":30649423,"title":"Species-specific detection of medically important aspergilli by a loop-mediated isothermal amplification method in chronic pulmonary aspergillosis","year":2019,"journal":"Medical Mycology","authors":"Kazuya Tone, Junko Suzuki, Mohamed Mahdi Alshahni, Kazuyoshi Kuwano, Koichi Makimura","doi":"10.1093\/mmy\/myy128","country":"","institute":"","deparment":"","abstract":"Chronic pulmonary aspergillosis (CPA) is a common subtype of pulmonary aspergillosis and a life-threatening disease. However, its diagnosis remains difficult due to the lack of specific clinical features and radiologic findings, as well as the difficulty of isolating Aspergillus spp. We developed a novel species-specific detection method of medically important aspergilli using a loop-mediated isothermal amplification (LAMP) for CPA. Specific LAMP primer sets for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, and Aspergillus nidulans were designed. The use of the LAMP assay was validated using respiratory specimens (CPA cases, n = 21; nonaspergillosis cases, n = 23). A total of 15 cases were positive in the CPA group (A. fumigatus, n = 5; A. flavus, n = 1; A. niger, n = 1; A. terreus, n = 7; A. nidulans, n = 1), but only three in the non-CPA group (A. niger, n = 2; A. terreus n = 1). The sensitivity and specificity of the diagnosis of CPA by the LAMP system were 71.4% and 87.0%, respectively. In conclusion, we developed a species-specific detection approach for five medically important aspergilli using the LAMP method. The system showed high sensitivity and specificity for diagnosis of CPA.","lamp_id":[{"id":"LPB00768","pathogen":"Aspergillus fumigatus","target":"TUB1"},{"id":"LPB00769","pathogen":"Aspergillus flavus","target":"BT"},{"id":"LPB00770","pathogen":"Aspergillus niger","target":"tub2"},{"id":"LPB00771","pathogen":"Aspergillus terreus","target":"TUB"},{"id":"LPB00772","pathogen":"Aspergillus nidulans","target":"beta-tubulin"}]},{"id":432,"pmid":31008450,"title":"Rapid and Accurate Diagnosis of the Respiratory Disease Pertussis on a Point-of-Care Biochip","year":2019,"journal":"eClinicalMedicine","authors":"Maowei Dou, Natalie Macias, Feng Shen, Jennifer Dien Bard, Delfina C Dom\u00ednguez, Xiujun Li","doi":"10.1016\/j.eclinm.2019.02.008","country":"","institute":"","deparment":"","abstract":"Background: Pertussis is a highly contagious respiratory disease caused by the bacterium Bordetella pertussis (B. pertussis). The infection is difficult to diagnose especially in underserved or resource-limited areas. We developed a low-cost and instrument-free diagnostic method for rapid and accurate detection of B. pertussis on a point-of-care (POC) testing device.\n\nMethods: We developed a paper\/polymer hybrid microfluidic biochip integrated with loop-mediated isothermal amplification (LAMP) method for the rapid and accurate detection of B. pertussis. This microfluidic approach was validated by testing 100 de-identified remnant clinical nasopharyngeal swabs and aspirates, which were confirmed to be either positive or negative for B. pertussis by a validated real-time PCR assay at the Children's Hospital Los Angeles.\n\nFindings: The instrument-free detection results could be successfully read by the naked eye within 45 min with a limit of detection (LOD) of 5 DNA copies per well. Our optimized bacterial lysis protocol allowed the direct testing of clinical samples without any complicated sample processing\/preparation (i.e. DNA extraction) or the use of any equipment (e.g. centrifuges). The validation of the microfluidic approach was accomplished by testing 100 clinical samples. High sensitivity (100%) and specificity (96%) with respect to real-time PCR were achieved.\n\nInterpretation: This microfluidic biochip shows great potential for point-of-care disease diagnosis in various venues including schools and physician's offices, especially in low-resource settings in developing nations.","lamp_id":[{"id":"LPB00773","pathogen":"Bordetella pertussis Tohama I","target":"PT promoter region"}]},{"id":433,"pmid":25187511,"title":"Loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia in HIV-uninfected immunocompromised patients with pulmonary infiltrates","year":2014,"journal":"Journal of Infection and Chemotherapy","authors":"Kei Nakashima, Masahiro Aoshima, Yoshihiro Ohkuni, Eri Hoshino, Kohei Hashimoto, Yoshihito Otsuka","doi":"10.1016\/j.jiac.2014.08.005","country":"","institute":"","deparment":"","abstract":"Loop-mediated isothermal ampli\ufb01cation (LAMP) is becoming an established nucleic acid ampli\ufb01cation method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. We retrospectively evaluated 78 consecutive HIV-uninfected patients who underwent LAMP method for diagnosing Pneumocystis pneumonia (PCP). Diagnosis of PCP was made by the detection of Pneumocystis jirovecii (P. jirovecii) with positive LAMP or conventional staining (CS) (Grocott methenamine silver staining or Diff-Quick\u2122) on the basis of compatible clinical symptoms and radiologic findings. Additionally, we reviewed HIV-uninfected immunocompromised patients who underwent subcontract PCR as a historical control. LAMP was positive in 10 (90.9%) of 11 positive-CS patients. Among 13 negative-CS patients with positive LAMP, 11 (84.6%) had PCP, and the remaining 2 were categorized as having P. jirovecii colonization. LDH levels in negative-CS PCP were higher than in positive-CS PCP (p = 0.026). (1 \u2192 3)-\u03b2-D-glucan levels in negative-CS PCP were lower than in positive-CS PCP (p = 0.011). The interval from symptom onset to diagnosis as PCP in LAMP group (3.45 \u00b1 1.77 days; n = 22) was shorter than in subcontract PCR group (6.90 \u00b1 2.28 days; n = 10; p < 0.001). As for patients without PCP, duration of unnecessary PCP treatment in LAMP group (2; 2-3 days; n = 10) was shorter than in subcontract PCR group (7; 7-12.25 days; n = 6; p = 0.003). LAMP showed higher sensitivity (95.4%) and positive predictive value (91.3%) than subcontract PCR did. Pneumocystis LAMP method is a sensitive and cost-effective diagnostic method and is easy to administer in general hospitals. In-house LAMP method would realize early diagnosis of PCP, resulting in improving PCP prognosis and reducing unnecessary PCP-specific treatment.","lamp_id":[{"id":"LPB00774","pathogen":"Pneumocystis jirovecii","target":"18S rRNA"}]},{"id":434,"pmid":17097200,"title":"Simple differentiation method of mumps Hoshino vaccine strain from wild strains by reverse transcription loop-mediated isothermal amplification (RT-LAMP)","year":2007,"journal":"Vaccine","authors":"Naoko Yoshida, Motoko Fujino, Yoshinori Ota, Tsugunori Notomi, Tetsuo Nakayama","doi":"10.1016\/j.vaccine.2006.09.093","country":"","institute":"","deparment":"","abstract":"Mumps virus is still circulating and annual mumps outbreaks occur with fluctuating magnitudes in Japan. Aseptic meningitis has been reported after vaccination and it would be of importance to determine whether this was related to the vaccination. The objective of this study was to develop a sensitive, specific and rapid diagnostic method for the differentiation of the Hoshino vaccine strain from circulating wild types. We developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method of the hemagglutinin neuraminidase (HN) region for the detection of mumps virus genome from clinical samples. The typical ladder pattern disappeared after the LAMP products of the Hoshino vaccine strain were digested with ScaI, but those of wild types were not cut by ScaI. We obtained 19 cerebro spinal fluids (CSF) from the patients with aseptic meningitis and 17 salivary swab samples from the patients with acute parotitis after mumps vaccination, in which one case was complicated with orchitis. Mumps virus genome was detected in 18 CSF samples and in all NPS by RT-LAMP. The Hoshino vaccine strain was identified in 16 out of 18 CSF RT-LAMP positives and in 11 out of 17 NPS samples and the remaining samples were identified as wild types. RT-LAMP followed by ScaI digestion is a sensitive, simple and rapid differential method and useful for laboratory surveillance for vaccine-adverse events.","lamp_id":[{"id":"LPB00775","pathogen":"Mumps virus","target":"HN"}]},{"id":435,"pmid":36466632,"title":"Development and clinical application of a endonuclease restriction real-time loop-mediated isothermal amplification (ERT-LAMP) assay for rapid detection of Haemophilus influenzae","year":2022,"journal":"Frontiers in Microbiology","authors":"Jinzhi Cheng, Yuhong Zhou, Xue Zhao, Jingrun Lu, Jiahong Wu, Yu Wang","doi":"10.3389\/fmicb.2022.1037343","country":"","institute":"","deparment":"","abstract":"Haemophilus influenzae is a main human pathogen that results in a series of diseases in children and adults, such as pneumonia, bacteremia, and meningitis. Although there are many detection methods, they cannot meet the requirements of an early diagnosis. For the prevention and control of H. influenzae infection, quick, sensitive, and particular diagnostics are crucial. Loop-mediated isothermal amplification (LAMP) coupled with restricted endonuclease digestion and real-time fluorescence (H. influenzae-ERT-LAMP) detection was employed to diagnose H. influenzae. H. influenzae-ERT-LAMP combines LAMP amplification, restriction endonuclease cleavage, and real-time fluorescence identification into a single-pot reaction, allowing for the rapid identification of H. influenzae in 40 min. The outer membrane protein (OMP) P6 gene of H. influenzae was employed to build a sequence of H. influenzae-ERT-LAMP primers. The limit of detection (LoD) of H. influenzae-ERT-LAMP test was 40 fg of genomic DNA per reaction, and the non-H. influenzae templates did not provide positive outcomes. To investigate the applicability of H. influenzae-ERT-LAMP method in clinical sample detection, 30 sputum specimens were obtained from individuals suspected of being infected with H. influenzae. H. influenzae-ERT-LAMP outcomes were in total agreement with LAMP-LFB and PCR. The H. influenzae-ERT-LAMP assay provides rapid, accurate, and sensitive detection making it a promising screening strategy in clinical and basic lab settings.","lamp_id":[{"id":"LPB00776","pathogen":"Haemophilus influenzae","target":"OMP P6"}]},{"id":436,"pmid":36385304,"title":"Direct capture and amplification of nucleic acids using a universal, elution-free magnetic bead-based method for rapid pathogen detection in multiple types of biological samples","year":2022,"journal":"Analytical and Bioanalytical Chemistry","authors":"Qianqian Jiang, Yang Li, Lin Huang, Jinling Guo, Ailin Wang, Cuiping Ma, Chao Shi","doi":"10.1007\/s00216-022-04422-8","country":"","institute":"","deparment":"","abstract":"Nucleic acid amplification tests (NAATs) have become an attractive approach for pathogen detection, and obtaining high-quality nucleic acid extracts from biological samples plays a critical role in ensuring accurate NAATs. In this work, we established an elution-free magnetic bead (MB)-based method by introducing polyethylene-polypropylene glycol (PEPPG) F68 in lysis buffer and using NaOH solution instead of alcohols as the washing buffer for rapid nucleic acid extraction from multiple types of biological samples, including nasopharyngeal swabs, serum, milk, and pork, which bypassed the nucleic acid elution step and allowed the nucleic acid\/MB composite to be directly used as the template for amplification reactions. The entire extraction process was able to be completed in approximately 7 min. Even though the nucleic acid\/MB composite could not be used for quantitative real-time PCR (qPCR) assays, this elution-free MB-based method significantly improved the sensitivity of the loop-mediated isothermal amplification (LAMP) assay. The sensitivity of the quantitative real-time LAMP (qLAMP) assays combined with this elution-free MB-based method showed an improvement of one to three orders of magnitude compared with qLAMP or qPCR assays combined with the traditional MB-based method. In addition to manual operation, like the traditional MB-based method, this universal, rapid, and facile nucleic acid extraction method also has potential for integration into automated robotic processing, making it particularly suitable for the establishment of an analysis platform for ultrafast and sensitive pathogen detection in various biological samples both in centralized laboratories and at remote sites.","lamp_id":[{"id":"LPB00777","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":437,"pmid":30042168,"title":"A multiplex loop-mediated isothermal amplification assay for rapid screening of Acinetobacter baumannii and D carbapenemase OXA-23 gene","year":2018,"journal":"Bioscience Reports","authors":"Rungong Yang, Honghong Zhang, Xiaoxia Li, Ling Ye, Meiliang Gong, Jinghui Yang, Jihong Yu, Jie Bai","doi":"10.1042\/BSR20180425","country":"","institute":"","deparment":"","abstract":"Background: Acinetobacter baumannii is a health burden responsible for various nosocomial infections, and bacteremia in particular. The resistance of A. baumannii to most antibiotics including carbapenem has increased. OXA-23-producing A. baumannii is the chief source of nosocomial outbreaks with carbapenem-resistant A. baumannii Successful antibiotic treatment relies on the accurate and rapid identification of infectious agents and drug resistance. Here, we describe a multiplex loop-mediated isothermal amplification (LAMP) assay for simultaneous and homogeneous identification for A. baumannii infection screening and drug-resistance gene detection. Methods: Four primer pairs were designed to amplify fragments of the recA gene of A. baumannii and the oxa-23 gene. The reaction with a 25 \u03bcl of final volume was performed at 63\u00b0C for 60 min. For comparative purposes, we used a traditional method of bacterial identification to evaluate assay efficacy. Results: The multiplex LAMP assay enables simultaneous and homogeneous detection of the recA gene of A. baumannii and the oxa-23 gene and requires less than 21 min with no pre-requisite for DNA purification prior to the amplification reaction. The detection is specific to A. baumannii, and the coincidence rate of the multiplex LAMP and the traditional method was 100%. Conclusions: Our data indicate that the multiplex LAMP assay is a rapid, sensitive, simultaneous and homogeneous method for screening of A. baumannii and its drug-resistance gene.","lamp_id":[{"id":"LPB00778","pathogen":"Acinetobacter baumannii","target":"recA"}]},{"id":438,"pmid":31164577,"title":"DNA Extraction with DNAzol and LAMP, Performed in a Heating Block as a Simple Procedure for Detection of Mycobacterium tuberculosis in Sputum Specimens","year":2018,"journal":"Methods and Protocols","authors":"\u00c1lvaro Rodr\u00edguez-Garc\u00eda, Rosa E. Mares, Patricia L. A. Mu\u00f1oz, Samuel G. Mel\u00e9ndez-L\u00f3pez, Alexei F. Licea-Navarro, Marco A. Ramos","doi":"10.3390\/mps1040037","country":"","institute":"","deparment":"","abstract":"Tuberculosis (TB) remains as a major public health issue in developing countries. Accurate detection is essential for the proper management of patients with active disease. Here, we present a simple DNAzol-LAMP (loop-mediated isothermal amplification) procedure for the detection of Mycobacterium tuberculosis in sputum specimens. Twenty smear-positive sputum samples were analyzed as follows: (i) Genetic material was extracted by a standard DNAzol protocol, and (ii) mycobacterial DNA was detected by a typical TB-specific loop-mediated isothermal amplification method. Results and diagnostic test performance attests to the suitability of the proposed procedure.","lamp_id":[{"id":"LPB00779","pathogen":"Mycobacterium tuberculosis H37Rv","target":"IS6110"}]},{"id":439,"pmid":25179393,"title":"Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria","year":2014,"journal":"Journal of Infection and Chemotherapy","authors":"Mitsuaki Nagasawa, Mitsuo Kaku, Kazunari Kamachi, Keigo Shibayama, Yoshichika Arakawa, Keizo Yamaguchi, Yoshikazu Ishii","doi":"10.1016\/j.jiac.2014.08.013","country":"","institute":"","deparment":"","abstract":"Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies\/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings.","lamp_id":[{"id":"LPB00780","pathogen":"Pseudomonas aeruginosa","target":"rmtA"},{"id":"LPB00781","pathogen":"Pseudomonas aeruginosa","target":"rmtB"},{"id":"LPB00784","pathogen":"Serratia marcescens","target":"armA"}]},{"id":440,"pmid":21047534,"title":"Efficacy of loop mediated isothermal amplification (LAMP) assay for the laboratory identification of Mycobacterium tuberculosis isolates in a resource limited setting","year":2011,"journal":"Journal of Microbiological Methods","authors":"G Geojith, S Dhanasekaran, Salesh P Chandran, John Kenneth","doi":"10.1016\/j.mimet.2010.10.015","country":"","institute":"","deparment":"","abstract":"Current methods of TB diagnosis are time consuming and less suited for developing countries. The LAMP (loop mediated isothermal amplification) is a rapid method more suitable for diagnosis in resource limited settings and has been proposed as a viable test requiring further evaluation for use as a laboratory method as well. We evaluated two LAMP assays, using culture lysates of clinical sputum samples (from Southern India) and compared it to a proprietary multiplex PCR reverse-hybridization line probe assay ('GenoType MTBC' from HAIN Lifescience GmbH, Germany). The LAMP procedure was modified to suit the local conditions. The Mycobacterium tuberculosis specific LAMP assay ('MTB LAMP') showed sensitivity and specificity, of 44.7% and 94.4% respectively in a 60 min format, 85.7% and 93.9% respectively in a 90 min format and 91.7%, and 90.9% respectively in a 120 min format. The Mycobacteria universal LAMP assay ('Muniv LAMP') showed a sensitivity of 99.1%. The LAMP was shown to be a rapid and accessible assay for the laboratory identification of M. tuberculosis isolates. Initial denaturation of template was shown to be essential for amplification in unpurified\/dilute samples and longer incubation was shown to increase the sensitivity. The need for modification of protocols to yield better efficacy in this scenario needs to be addressed in subsequent studies.","lamp_id":[{"id":"LPB00782","pathogen":"Mycobacterium tuberculosis H37Rv","target":"rimM"},{"id":"LPB00783","pathogen":"Mycobacterium avium","target":"gyrB"}]},{"id":441,"pmid":35213596,"title":"Sample-to-answer, extraction-free, real-time RT-LAMP test for SARS-CoV-2 in nasopharyngeal, nasal, and saliva samples: Implications and use for surveillance testing","year":2022,"journal":"PLoS One","authors":"Kathryn A Kundrod, Mary E Natoli, Megan M Chang, Chelsey A Smith, Sai Paul, Dereq Ogoe, Christopher Goh, Akshaya Santhanaraj, Anthony Price, Karen W Eldin, Keyur P Patel, Ellen Baker, Kathleen M Schmeler, Rebecca Richards-Kortum","doi":"10.1371\/journal.pone.0264130","country":"","institute":"","deparment":"","abstract":"The global COVID-19 pandemic has highlighted the need for rapid, accurate and accessible nucleic acid tests to enable timely identification of infected individuals. We optimized a sample-to-answer nucleic acid test for SARS-CoV-2 that provides results in <1 hour using inexpensive and readily available reagents. The test workflow includes a simple lysis and viral inactivation protocol followed by direct isothermal amplification of viral RNA using RT-LAMP. The assay was validated using two different instruments, a portable isothermal fluorimeter and a standard thermocycler. Results of the RT-LAMP assay were compared to traditional RT-qPCR for nasopharyngeal swabs, nasal swabs, and saliva collected from a cohort of patients hospitalized due to COVID-19. For all three sample types, positive agreement with RT-LAMP performed using the isothermal fluorimeter was 100% for samples with Ct <30 and 69-91% for samples with Ct <40. Following validation, the test was successfully scaled to test the saliva of up to 400 asymptomatic individuals per day as part of the campus surveillance program at Rice University. Successful development, validation, and scaling of this sample-to-answer, extraction-free real-time RT-LAMP test for SARS-CoV-2 adds a highly adaptable tool to efforts to control the COVID-19 pandemic, and can inform test development strategies for future infectious disease threats.","lamp_id":[{"id":"LPB00785","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00786","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00787","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00814","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00815","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00816","pathogen":"SARS-CoV-2","target":"ORF1a"}]},{"id":442,"pmid":32754129,"title":"Rapid Detection of mecA and femA Genes by Loop-Mediated Isothermal Amplification in a Microfluidic System for Discrimination of Different Staphylococcal Species and Prediction of Methicillin Resistance","year":2020,"journal":"Frontiers in Microbiology","authors":"Xiangrui Meng, Guohao Zhang, Bo Sun, Shujun Liu, Yadong Wang, Ming Gao, Yubo Fan, Guojun Zhang, Guangzhi Shi, Xixiong Kang","doi":"10.3389\/fmicb.2020.01487","country":"","institute":"","deparment":"","abstract":"Staphylococcal infection is one of the most pressing problems in modern medicine due to the increasing antibiotic resistance with the overuse of antibiotics. Conventional methods for identification and antimicrobial susceptibility testing (AST) generally take 3-7 days and require skilled technicians. In this study, a microfluidic device based on loop-mediated isothermal amplification (LAMP) was developed, which could discriminate Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis and predict their methicillin resistance by targeting the mecA and femA genes within 70 min including the hands-on time. Multiplex and real-time detection was achieved in a closed system without aerosol contamination. The limits of detection (LODs) for S. aureus, S. epidermidis, S. hominis, and methicillin-resistant S. aureus (MRSA) were 20 CFU\/reaction, while that for S. haemolyticus was 200 CFU\/reaction. A total of 102 positive cultures of cerebrospinal fluid (CSF) were also tested, and the results were in good agreement with those from conventional methods. Furthermore, mixed cultures were readily identified by our method. The portable and integrated device is rapid, accurate, and easy to use, which can provide information for prompt institution of proper antimicrobial therapy and has great potential for clinical applications, especially in resource-limited settings.","lamp_id":[{"id":"LPB00788","pathogen":"Staphylococcus aureus","target":"mecA"},{"id":"LPB00789","pathogen":"Staphylococcus aureus","target":"femA"}]},{"id":443,"pmid":30404341,"title":"Rapid Capture and Analysis of Airborne Staphylococcus aureus in the Hospital Using a Microfluidic Chip","year":2016,"journal":"Micromachines","authors":"Xiran Jiang, Yingchao Liu, Qi Liu, Wenwen Jing, Kairong Qin, Guodong Sui","doi":"10.3390\/mi7090169","country":"","institute":"","deparment":"","abstract":"In this study we developed a microfluidic chip for the rapid capture, enrichment and detection of airborne Staphylococcus (S.) aureus. The whole analysis took about 4 h and 40 min from airborne sample collection to loop-mediated isothermal amplification (LAMP), with a detection limit down to about 27 cells. The process did not require DNA purification. The chip was validated using standard bacteria bioaerosol and was directly used for clinical airborne pathogen sampling in hospital settings. This is the first report on the capture and analysis of airborne S. aureus using a novel microfluidic technique, a process that could have a very promising platform for hospital airborne infection prevention (HAIP).","lamp_id":[{"id":"LPB00790","pathogen":"Staphylococcus aureus","target":"nuc"}]},{"id":444,"pmid":28578093,"title":"Direct bacterial loop-mediated isothermal amplification detection on the pathogenic features of the nosocomial pathogen - Methicillin resistant Staphylococcus aureus strains with respiratory origins","year":2017,"journal":"Microbial Pathogenesis","authors":"Qun Lin, Pusheng Xu, Jiaowu Li, Yin Chen, Jieyi Feng","doi":"10.1016\/j.micpath.2017.05.044","country":"","institute":"","deparment":"","abstract":"Loop-mediated isothermal amplification based detection assays using bacterial culture or colony for direct detection of methicillin resistant Staphylococcus aureus(MRSA) had been developed and evaluated, followed by its extensive application on a large scale of clinical MRSA isolated from respiratory origins, including nasal swabs and sputums. Six primers, including outer primers, inner primers and loop primers, were specifically designed for recognizing eight distinct sequences on four targets: 16SrRNA, femA, mecA and orfX. Twenty-seven reference strains were used to develop, evaluate and optimize this assay. Then, a total of 532 clinical MRSA isolates were employed for each detected targets. And the results were determined through both visual observation of the color change by naked eye and electrophoresis. The specific of each primer had been confirmed, and the optimal amplification was obtained under 65 \u00b0C for 40 min. The limit of detections (LOD) of bacteria culture LAMP assays were determined to be 10^4 CFU\/ml for 16S rRNA, femA, as well as orfX and 10^5 CFU\/ml for mecA, respectively. The established novel assays on MRSA detection may provide new strategies for rapid detection of foodborne pathogens.","lamp_id":[{"id":"LPB00791","pathogen":"Staphylococcus aureus","target":"16S rRNA"},{"id":"LPB00792","pathogen":"Staphylococcus aureus","target":"orfX"},{"id":"LPB00793","pathogen":"Staphylococcus aureus","target":"mecA"},{"id":"LPB00794","pathogen":"Staphylococcus aureus","target":"femA"}]},{"id":445,"pmid":36061848,"title":"Feasibility of Loop-Mediated Isothermal Amplification for Rapid Detection of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus in Tissue Samples","year":2022,"journal":"Clinics in Orthopedic Surgery","authors":"Sang-Gyun Kim, Gi Won Choi, Won Seok Choi, Chae Seung Lim, Woong Sik Jang, Ji Hoon Bae","doi":"10.4055\/cios21277","country":"","institute":"","deparment":"","abstract":"Background: To date, few studies have investigated the feasibility of the loop-mediated isothermal amplification (LAMP) assay for identifying pathogens in tissue samples. This study aimed to investigate the feasibility of LAMP for the rapid detection of methicillin-susceptible or methicillin-resistant Staphylococcus aureus (MSSA or MRSA) in tissue samples, using a bead-beating DNA extraction method.\n\nMethods: Twenty tissue samples infected with either MSSA (n = 10) or MRSA (n = 10) were obtained from patients who underwent orthopedic surgery for suspected musculoskeletal infection between December 2019 and September 2020. DNA was extracted from the infected tissue samples using the bead-beating method. A multiplex LAMP assay was conducted to identify MSSA and MRSA infections. To recognize the Staphylococcus genus, S. aureus, and methicillin resistance, 3 sets of 6 primers for the 16S ribosomal ribonucleic acid (rRNA) and the femA and mecA genes were used, respectively. The limit of detection and sensitivity (detection rate) of the LAMP assay for diagnosing MSSA and MRSA infection were analyzed.\n\nResults: The LAMP result was positive for samples containing 10^3 colony-forming unit (CFU)\/mL for 16S rRNA, 10^4 CFU\/mL for femA, and 10^5 CFU\/mL for mecA. The limits of detection for 16S rRNA and femA were not different between MSSA and MRSA. For the 10 MSSA-positive samples, the LAMP assay showed 100% positive reactions for 16S rRNA and femA and a 100% negative reaction for mecA. For the 10 MRSA-positive samples, the LAMP assay showed 100% positive reactions for 16S rRNA and mecA but only 90% positive reactions for femA. The sensitivity (detection rate) of the LAMP assay for identifying MSSA and MRSA in infected tissue samples was 100% and 90%, respectively.\n\nConclusions: The results of this study suggest that the LAMP assay performed with tissue DNA samples can be a useful diagnostic method for the rapid detection of musculoskeletal infections caused by MSSA and MRSA.","lamp_id":[{"id":"LPB00795","pathogen":"Staphylococcus aureus","target":"femA"},{"id":"LPB00796","pathogen":"Staphylococcus aureus","target":"16S rRNA"},{"id":"LPB00797","pathogen":"Staphylococcus aureus","target":"mecA"}]},{"id":446,"pmid":33439634,"title":"Fluorometric Paper-Based, Loop-Mediated Isothermal Amplification Devices for Quantitative Point-of-Care Detection of Methicillin-Resistant Staphylococcus aureus (MRSA)","year":2021,"journal":"ACS Sensors","authors":"Ilada Choopara, Akkapol Suea-Ngam, Yothin Teethaisong, Philip D Howes, Mathias Schmelcher, Asada Leelahavanichkul, Sudaluck Thunyaharn, Doonyapong Wongsawaeng, Andrew J deMello, Deborah Dean, Naraporn Somboonna","doi":"10.1021\/acssensors.0c01405","country":"","institute":"","deparment":"","abstract":"Loop-mediated isothermal amplification (LAMP) has been widely used to detect many infectious diseases. However, minor inconveniences during the steps of adding reaction ingredients and lack of simple color results hinder point-of-care detection. We therefore invented a fluorometric paper-based LAMP by incorporating LAMP reagents, including a biotinylated primer, onto a cellulose membrane paper, with a simple DNA fluorescent dye incubation that demonstrated rapid and accurate results parallel to quantitative polymerase chain reaction (qPCR) methods. This technology allows for instant paper strip detection of methicillin-resistant Staphylococcus aureus (MRSA) in the laboratory and clinical samples. MRSA represents a major public health problem as it can cause infections in different parts of the human body and yet is resistant to commonly used antibiotics. In this study, we optimized LAMP reaction ingredients and incubation conditions following a central composite design (CCD) that yielded the shortest reaction time with high sensitivity. These CCD components and conditions were used to construct the paper-based LAMP reaction by immobilizing the biotinylated primer and the rest of the LAMP reagents to produce the ready-to-use MRSA diagnostic device. Our paper-based LAMP device could detect as low as 10 ag (equivalent to 1 copy) of the MRSA gene mecA within 36-43 min, was evaluated using both laboratory (individual cultures of MRSA and non-MRSA bacteria) and clinical blood samples to be 100% specific and sensitive compared to qPCR results, and had 35 day stability under 25 \u00b0C storage. Furthermore, the color readout allows for quantitation of MRSA copies. Hence, this device is applicable for point-of-care MRSA detection.","lamp_id":[{"id":"LPB00798","pathogen":"Staphylococcus aureus","target":"mecA"}]},{"id":447,"pmid":17593498,"title":"Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures","year":2007,"journal":"Journal of Infection and Chemotherapy","authors":"Yoshiki Misawa, Atsushi Yoshida, Ryoichi Saito, Honami Yoshida, Katsuko Okuzumi, Nobue Ito, Mitsumasa Okada, Kyoji Moriya, Kazuhiko Koike","doi":"10.1007\/s10156-007-0508-9","country":"","institute":"","deparment":"","abstract":"Staphylococcus aureus is the most important pathogen in nosocomial infections, including bloodstream infections. Prompt identification of S. aureus from blood cultures and detection of methicillin resistance are essential in cases of suspected sepsis. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63 degrees C) with high specificity, efficiency, and rapidity, was applied to detect methicillin-resistant S. aureus (MRSA) directly from positive blood culture bottles. MRSA-LAMP, which targets the spa gene, encoding S. aureus-specific protein A, and the mecA gene, encoding penicillin-binding protein-2' for methicillin resistance, could detect MRSA within 2 h after the blood culture signal became positive. The diagnostic values of LAMP, compared to a duplex real-time polymerase chain reaction (Drt-PCR) assay, were 92.3% and 96.2% sensitivity, 100% and 100% specificity, 100% and 100% positive predictive value (PPV), and 96.9% and 98.4% negative predictive value (NPV), respectively. These two methods had almost the same results, but the LAMP method is more cost-effective and provides excellent availability for rapid examination in a hospital clinical laboratory. Therefore, the LAMP assay appears to be a sensitive and reliable new method to diagnose MRSA bloodstream infection for appropriate antibiotic therapy.","lamp_id":[{"id":"LPB00799","pathogen":"Staphylococcus aureus","target":"spa"},{"id":"LPB00800","pathogen":"Staphylococcus aureus","target":"mecA"}]},{"id":448,"pmid":36143901,"title":"Developing a Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Seven Respiratory Viruses including SARS-CoV-2","year":2022,"journal":"Medicina","authors":"Min-Young Lee, Vu-Minh Phan, Woo-In Lee, Yee-Hyung Kim, Sung-Wook Kang, Tae-Seok Seo","doi":"10.3390\/medicina58091224","country":"","institute":"","deparment":"","abstract":"Background and Objectives: The coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to be a pandemic even in 2022. As the initial symptoms of COVID-19 overlap with those of infections from other respiratory viruses, an accurate and rapid diagnosis of COVID-19 is essential for administering appropriate treatment to patients. Currently, the most widely used method for detecting respiratory viruses is based on real-time polymerase chain reaction (PCR) and includes reverse-transcription real-time quantitative PCR (RT-qPCR). However, RT-qPCR assays require sophisticated facilities and are time-consuming. This study aimed to develop a real-time quantitative loop-mediated isothermal amplification (RT-qLAMP) assay and compare its analytical performance with RT-qPCR. Materials and Methods: A total of 315 nasopharyngeal swabs from patients with symptoms of respiratory infections were included in this study. A primary screening of the specimens was performed using RT-qPCR. RNA\/DNA from standard strains for respiratory viruses and heat-inactivated preparations of standard strains for SARS-CoV-2 were used to evaluate the accuracy and target specificity of the RT-qLAMP assay. Results: We successfully developed an RT-qLAMP assay for seven respiratory viruses: respiratory syncytial virus (RSV) A, RSV B, adenovirus, influenza (Flu) A (H1N1 and H3N2), Flu B, and SARS-CoV-2. RT-qLAMP was performed in a final reaction volume of 9.6 \u00b5L. No cross-reactivity was observed. Compared with the RT-PCR results, the sensitivity and specificity of the RT-qLAMP assay were 95.1% and 100%, respectively. The agreement between the two methods was 97.1%. The median amplification time to RT-qLAMP positivity was 22:34 min (range: 6:80-47:98 min). Conclusions: The RT-qLAMP assay requires a small number of reagents and samples and is performed with an isothermal reaction. This study established a fast, simple, and sensitive test that can be applied to point-of-care testing devices to facilitate the detection of respiratory viruses, including SARS-CoV-2.","lamp_id":[{"id":"LPB00801","pathogen":"HAdV","target":null},{"id":"LPB00802","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00803","pathogen":"Influenza A virus (H1N1)","target":"HA"},{"id":"LPB00804","pathogen":"Influenza A virus (H3N2)","target":"HA"},{"id":"LPB00805","pathogen":"Influenza A virus (H3N2)","target":"HA"},{"id":"LPB00806","pathogen":"RSV A","target":null},{"id":"LPB00807","pathogen":"RSV B","target":null},{"id":"LPB00808","pathogen":"Influenza B virus","target":null},{"id":"LPB00809","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"},{"id":"LPB00810","pathogen":"SARS-CoV-2","target":"S"}]},{"id":449,"pmid":34250011,"title":"Development of Loop-Mediated Isothermal Amplification Assay for Detection of Clinically Significant Members of Acinetobacter calcoaceticus-baumannii Complex and Associated Carbapenem Resistance","year":2021,"journal":"Frontiers in Molecular Biosciences","authors":"Amit Sharma, Rajni Gaind","doi":"10.3389\/fmolb.2021.659256","country":"","institute":"","deparment":"","abstract":"Background: Acinetobacter calcoaceticus-baumannii (ACB) complex has emerged as an important nosocomial pathogen and is associated with life-threatening infections, especially among ICU patients, including neonates. Carbapenem resistance in Acinetobacter baumannii has emerged globally and is commonly mediated by bla OXA-23. Clinically significant infections with carbapenem-resistant Acinetobacter baumannii (CRAB) are a major concern since therapeutic options are limited and associated mortality is high. Early diagnosis of both the pathogen and resistance is important to initiate the optimal therapy and prevent selection of resistance. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of the ACB complex and carbapenem resistance mediated by bla OXA-23. Methodology: Universal LAMP primers were designed for the detection of significant members of the ACB complex and carbapenem resistance targeting the ITS 16S-23S rRNA and bla OXA-23 gene respectively. The optimal conditions for the LAMP assay were standardized for each primer set using standard ATCC strains. The sensitivity of the LAMP assay was assessed based on the limit of detection (LOD) using different DNA concentrations and colony counts. The specificity of LAMP was determined using the non-ACB complex and non-Acinetobacter species. The results of the LAMP assay were compared with those of polymerase chain reaction (PCR). Results: The optimal temperature for the LAMP assay was 65\u00b0C, and the detection time varied with various primers designed. Using the ITS Ab1 primer, LODs of LAMP and PCR assays were 100 pg\/\u03bcl and 1 ng\/\u03bcl of DNA concentration and 10^4 cfu\/ml and 10^8 cfu\/ml of colony count, respectively. The LAMP assay was 10- and 10^4-fold more sensitive than PCR using DNA concentration and colony count, respectively. The LAMP assay was found to be specific for clinically important ACB complex species.","lamp_id":[{"id":"LPB00811","pathogen":"Acinetobacter baumannii","target":"16S\u201323S rRNA"},{"id":"LPB00812","pathogen":"Acinetobacter baumannii","target":"16S\u201323S rRNA"},{"id":"LPB00813","pathogen":"Acinetobacter baumannii","target":"bla OXA-23"}]},{"id":450,"pmid":20149082,"title":"Rapid detection of mecA and spa by the loop-mediated isothermal amplification (LAMP) method","year":2010,"journal":"Letters in Applied Microbiology","authors":"Y Koide, H Maeda, K Yamabe, K Naruishi, T Yamamoto, S Kokeguchi, S Takashiba","doi":"10.1111\/j.1472-765X.2010.02806.x","country":"","institute":"","deparment":"","abstract":"Aim: To develop a detection assay for staphylococcal mecA and spa by using loop-mediated isothermal amplification (LAMP) method.\n\nMethods and results: Staphylococcus aureus and other related species were subjected to the detection of mecA and spa by both PCR and LAMP methods. The LAMP successfully amplified the genes under isothermal conditions at 64 degrees C within 60 min, and demonstrated identical results with the conventional PCR methods. The detection limits of the LAMP for mecA and spa, by gel electrophoresis, were 10(2) and 10 cells per tube, respectively. The naked-eye inspections were possible with 10(3) and 10 cells for detection of mecA and spa, respectively. The LAMP method was then applied to sputum and dental plaque samples. The LAMP and PCR demonstrated identical results for the plaque samples, although frequency in detection of mecA and spa by the LAMP was relatively lower for the sputum samples when compared to the PCR methods.\n\nConclusion: Application of the LAMP enabled a rapid detection assay for mecA and spa. The assay may be applicable to clinical plaque samples.","lamp_id":[{"id":"LPB00818","pathogen":"Staphylococcus aureus","target":"spa"},{"id":"LPB00819","pathogen":"Staphylococcus aureus","target":"mecA"}]},{"id":451,"pmid":34967203,"title":"Membrane-Based In-Gel Loop-Mediated Isothermal Amplification (mgLAMP) System for SARS-CoV-2 Quantification in Environmental Waters","year":2021,"journal":"Environmental Science & Technology","authors":"Yanzhe Zhu, Xunyi Wu, Alan Gu, Leopold Dobelle, Cl\u00e9ment A. Cid, Jing Li, Michael R. Hoffmann","doi":"10.1021\/acs.est.1c04623","country":"","institute":"","deparment":"","abstract":"Since the COVID-19 pandemic is expected to become endemic, quantification of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in ambient waters is critical for environmental surveillance and for early detection of outbreaks. Herein, we report the development of a membrane-based in-gel loop-mediated isothermal amplification (mgLAMP) system that is designed for the rapid point-of-use quantification of SARS-CoV-2 particles in environmental waters. The mgLAMP system integrates the viral concentration, in-assay viral lysis, and on-membrane hydrogel-based RT-LAMP quantification using enhanced fluorescence detection with a target-specific probe. With a sample-to-result time of less than 1 h, mgLAMP successfully detected SARS-CoV-2 below 0.96 copies\/mL in Milli-Q water. In surface water, the lowest detected SARS-CoV-2 concentration was 93 copies\/mL for mgLAMP, while the reverse transcription quantitative polymerase chain reaction (RT-qPCR) with optimal pretreatment was inhibited at 930 copies\/mL. A 3D-printed portable device is designed to integrate heated incubation and fluorescence illumination for the simultaneous analysis of nine mgLAMP assays. Smartphone-based imaging and machine learning-based image processing are used for the interpretation of results. In this report, we demonstrate that mgLAMP is a promising method for large-scale environmental surveillance of SARS-CoV-2 without the need for specialized equipment, highly trained personnel, and labor-intensive procedures.","lamp_id":[{"id":"LPB00820","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00821","pathogen":"SARS-CoV-2","target":"ORF1ab (NSP3)"}]},{"id":452,"pmid":32431523,"title":"Nanoparticles-Based Biosensor Coupled with Multiplex Loop-Mediated Isothermal Amplification for Detection of Staphylococcus aureus and Identification of Methicillin-Resistant S. aureus","year":2020,"journal":"Infection and Drug Resistance","authors":"Luxi Jiang, Xiaomeng Li, Rumeng Gu, Deguang Mu","doi":"10.2147\/IDR.S243881","country":"","institute":"","deparment":"","abstract":"Introduction: Staphylococcus aureus (S. aureus), including methicillin-resistant S. aureus (MRSA), is a common human pathogen, which can cause a variety of infections from mild to severe. In this article, a new diagnostic method called multiplex loop-mediated isothermal amplification combined with nanoparticles-based lateral flow biosensor (mLAMP-LFB) has been developed, which was proved to be fast, reliable, and simple for detecting S. aureus, and differentiate MRSA from methicillin-susceptible S. aureus (MSSA).\n\nMaterials and methods: We designed a set of six primers targeting the nuc gene of S. aureus, and a set of five primers targeting the mecA gene of MRSA. The lateral flow biosensor visually reported the S. aureus-LAMP results within 2 mins. S. aureus species and non-S. aureus species were used to identify the specificity and sensitivity of the assay.\n\nResults: The best conditions for LAMP were 50 mins at 63\u00b0C, and the sensitivity was 100 fg. No cross-reactivity was shown and the specificity of this assay is 100%. This assay requires 20 mins for DNA preparation, 50 mins for isothermal amplification and 2 mins for biosensor detection. The total time is within 75 mins. Among 96 sputum samples, LAMP-LFB and traditional culture method showed the same results, 8 (8.33%) samples were MRSA-positive, and 9 (9.38%) samples were MSSA-positive. Seven (7.29%) samples were MRSA-positive and 7 (7.29%) were MSSA-positive by PCR method. Compared with the culture method, diagnostic accuracy of m-LAMP-LFB assay was 100%. The results showed that the m-LAMP-LFB method has better detection ability than the PCR method.\n\nDiscussion: In short, this m-LAMP-LFB assay is a specific and sensitive method that can quickly identify S. aureus stains, and distinguish MRSA from MSSA, and can be used as a new molecular method for detection of S. aureus in laboratories.","lamp_id":[{"id":"LPB00822","pathogen":"Staphylococcus aureus","target":"nuc"},{"id":"LPB00823","pathogen":"Staphylococcus aureus","target":"mecA"}]},{"id":453,"pmid":23509796,"title":"Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Staphylococcus aureus","year":2013,"journal":"BioMed Research International","authors":"King Ting Lim, Cindy Shuan Ju Teh, Kwai Lin Thong","doi":"10.1155\/2013\/895816","country":"","institute":"","deparment":"","abstract":"Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is an important human pathogen that produces a variety of toxins and causes a wide range of infections, including soft-tissue infections, bacteremia, and staphylococcal food poisoning. A loop-mediated isothermal amplification (LAMP) assay targeting the arcC gene of S. aureus was developed and evaluated with 119 S. aureus and 25 non-S. aureus strains. The usefulness of the assay was compared with the PCR method that targets spa and arcC genes. The optimal temperature for the LAMP assay was 58.5\u00b0C with a detection limit of 2.5 ng\/\u03bcL and 10(2) CFU\/mL when compared to 12.5 ng\/\u03bcL and 10(3) CFU\/mL for PCR (spa and arcC). Both LAMP and PCR assays were 100% specific, 100% sensitive, 100% positive predictive value (PPV), and 100% negative predictive value (NPV). When tested on 30 spiked blood specimens (21 MRSA, eight non-S. aureus and one negative control), the performance of LAMP and PCR was comparable: 100% specific, 100% sensitive, 100% PPV, and 100% NPV. In conclusion, the LAMP assay was equally specific with a shorter detection time when compared to PCR in the identification of S. aureus. The LAMP assay is a promising alternative method for the rapid identification of S. aureus and could be used in resource-limited laboratories and fields.","lamp_id":[{"id":"LPB00824","pathogen":"Staphylococcus aureus","target":"arcC"}]},{"id":454,"pmid":26159153,"title":"SPR-DNA array for detection of methicillin-resistant Staphylococcus aureus (MRSA) in combination with loop-mediated isothermal amplification","year":2015,"journal":"Biosensors and Bioelectronics","authors":"Kawin Nawattanapaiboon, Wansika Kiatpathomchai, Pitak Santanirand, Apirom Vongsakulyanon, Ratthasart Amarit, Armote Somboonkaew, Boonsong Sutapun, Toemsak Srikhirin","doi":"10.1016\/j.bios.2015.06.038","country":"","institute":"","deparment":"","abstract":"In this study, we evaluated surface plasmon resonance imaging (SPR imaging) as a DNA biosensor for the detection of methicillin-resistant Staphylococcus aureus (MRSA) which is one of the most common causes of nosocomial infections. The DNA sample were collected from clinical specimens, including sputum and blood hemoculture were undergone LAMP amplification for 0.18 kbp and 0.23 kbp DNA fragments of femB and mecA genes, respectively. The self-assembled monolayer surface (SAMs) was used for immobilized streptavidin-biotinylated probes on the sensor surface for the detection of LAMP amplicons from MRSA. Both LAMP amplicons were simultaneously hybridized with ssDNA probes immobilized onto a bio-functionalized surface to detect specific targets in the multiplex DNA array platform. In addition, the sensor surface could be regenerated allowing at least five cycles of use with a shortened assay time. The detection limit of LAMP-SPR sensing was 10 copies\/\u00b5l and LAMP-SPR sensing system showed a good selectivity toward the MRSA.","lamp_id":[{"id":"LPB00825","pathogen":"Staphylococcus aureus","target":"femB"},{"id":"LPB00826","pathogen":"Staphylococcus aureus","target":"mecA"}]},{"id":455,"pmid":27607914,"title":"A rapid loop-mediated isothermal amplification (LAMP) method for detection of the macrolide-streptogramin type B resistance gene msrA in Staphylococcus aureus","year":2016,"journal":"Journal of Global Antimicrobial Resistance","authors":"Xiao-Qin Mu, Bin-Bin Liu, Ephraim Hui, William Huang, Li-Chen Yao, Li-Bo Duo, Wen-Ying Sun, Gui-Qiu Li, Fu-Xiang Wang, Shu-Lin Liu","doi":"10.1016\/j.jgar.2016.07.006","country":"","institute":"","deparment":"","abstract":"Macrolide-streptogramin type B resistance (the MSB phenotype) is a multidrug resistance phenotype in Staphylococcus aureus conferred by the resistance gene msrA. However, bacteria having the MSB phenotype are susceptible to lincosamides and 16-membered ring macrolides, which makes profiling resistance genes necessary and urgent for timely and appropriate use of antimicrobials. In this study, the loop-mediated isothermal amplification (LAMP) assay was optimized for prompt detection of the msrA gene. msrA gene sequences were obtained from the National Center for Biotechnology Information (NCBI) database and primers were designed using the LAMP primer designing software PrimerExplorer v.4, which together recognize seven distinct regions of the msrA gene. The specific LAMP primer set designed in this study could amplify the msrA gene within 25min at an isothermal temperature of 62\u00b0C. More importantly, the msrA gene could be detected at a sensitivity as low as 100pg. Furthermore, this optimized LAMP assay provided swift detection of the msrA gene even directly from human specimens. In conclusion, this assay may have great clinical application potential for detection of the msrA gene.","lamp_id":[{"id":"LPB00827","pathogen":"Staphylococcus aureus","target":"msrA"}]},{"id":456,"pmid":null,"title":"Surface detection of SARS-CoV-2 by lateral flow LAMP","year":2022,"journal":"bioRxiv","authors":"Isabelle Dahl Acker, Mark Joseph Ware, John Russell Bracht","doi":"10.1101\/2022.04.04.487067","country":"","institute":"","deparment":"","abstract":"Slowing the transmission of SARS-CoV-2 requires rapid and accurate diagnostic testing. Toward this end, loop-mediated isothermal amplification (LAMP), an isothermal genomic detection method, offers great promise but the readout tends to be difficult because it does not generate linear DNA products. Rapid antigen tests are coupled to lateral flow strips, with one (negative) or two (positive) bands providing simple rapid readout, but are less sensitive than genomic amplification methods. To address the need for a genomic amplification method that can be visualized on a lateral flow strip, we developed a novel strand-displacement probe. In this work we validate this pipeline for purified RNA, intact virus, and even virus deposited onto a surface. We demonstrate robust sensitivity (100 genomic copies) and and we demonstrate the utility of our assay as a surveillance system, with the capability to detect viral particles from surfaces, even after a week of complete dry-down. Our innovation couples the diagnostic advantages of a nucleic acid amplification test (NAAT) with the simplicity of lateral-flow readouts.","lamp_id":[{"id":"LPB00828","pathogen":"SARS-CoV-2","target":"N"}]},{"id":457,"pmid":34763467,"title":"Rapid detection of multiple pathogens by the combined loop-mediated isothermal amplification technology and microfluidic chip technology","year":2021,"journal":"Annals of Palliative Medicine","authors":"Hongling Ou, Yan Wang, Qiyuan Wang, Yingying Ma, Chen Liu, Lianling Jia, Qiaoyun Zhang, Mengwen Li, Xinxin Feng, Meng Li, Xinru Wang, Chengbin Wang","doi":"10.21037\/apm-21-2792","country":"","institute":"","deparment":"","abstract":"Background: A rapid detection system for multiple pathogens that combines loop-mediated isothermal amplification (LAMP) technology and microfluidic chip technology was established.\n\nMethods: Primers were designed for the specific conservative genes of the multiple pathogens. A rapid detection method for multiple pathogens based on LAMP technology was established using the primer screening and optimization of reaction conditions. The microfluidic chip was designed. manufactured, and combined with the LAMP method for detection pathogenic bacteria as detected by a chip detector. After this, the detection limit and anti-interference ability of the chip were evaluated, and the accuracy of chip was verified by clinical samples.\n\nResults: A rapid microfluidic detection system for Staphylococcus aureus, Escherichia coli, Pneumoniae klebsiella, Shigella, Methicillin-resistant Staphylococcus Aureus (MRSA) and Candida albicans was established. The detection limits of the 6 strains above were 6.95, 44.6, 3.89, 15.33, 16.45, and 463 pg\/\u00b5L, respectively; there was no cross-reaction with the other 15 strains; analysis of ROC curve showed the best cut-off values for the 6 strains are 38.5, 21.25, 31.5, 36.5, 22.5 and 33.75 respectively and the area under the curve for the 6 strains was 0.91, 0.91, 0.83, 0.97, 0.96, and 0.9, respectively, as analyzed by receiver operating characteristic curve (ROC). A total of 278 clinical samples collected including blood, urine, sputum and drainage fluid were analyzed, and the total coincidence rates were 0.91, 0.83, 0.75, 0.99, 0.92 and 0.76, respectively.\n\nConclusions: The establishment of microfluidic detection chips for multiple pathogen types can be used for the rapid detection of bacteria and may be particularly useful in environments with the limited equipment and personnel, such as infection sites, grassroots hospitals, disaster area rescue, etc.","lamp_id":[{"id":"LPB00829","pathogen":"Staphylococcus aureus","target":"mecA"},{"id":"LPB00830","pathogen":"Staphylococcus aureus","target":"nuc"},{"id":"LPB00831","pathogen":"Klebsiella pneumoniae","target":"fimD"}]},{"id":458,"pmid":33251714,"title":"In Situ Nucleic Acid Amplification and Ultrasensitive Colorimetric Readout in a Paper-Based Analytical Device Using Silver Nanoplates","year":2020,"journal":"Advanced Healthcare Materials","authors":"Akkapol Suea-Ngam, Ilada Choopara, Shangkun Li, Mathias Schmelcher, Naraporn Somboonna, Philip D Howes, Andrew J deMello","doi":"10.1002\/adhm.202001755","country":"","institute":"","deparment":"","abstract":"A rapid, highly sensitive, and quantitative colorimetric paper-based analytical device (PAD) based on silver nanoplates (AgNPls) and loop-mediated isothermal amplification (LAMP) is presented. It is shown that cauliflower-like concatemer LAMP products can mediate crystal etching of AgNPls, with a threefold signal enhancement versus linear dsDNA. Methicillin-resistant Staphylococcus aureus (MRSA), an antimicrobial resistant bacterium that poses a formidable risk with persistently high mortality, is used as a model pathogen. Due to the excellent color contrast provided by AgNPls, the PAD allows qualitative analysis by the naked eye and quantitative analysis using a smartphone camera, with detection limits down to a single copy in just 30 min, and a linear response from 1 to 10^4 copies (R^2 = 0.994). The entire assay runs in situ on the paper surface, which drastically simplifies operation of the device. This is the first demonstration of single copy detection using a colorimetric readout, and the developed PAD shows great promise for translation into an ultrasensitive gene-based point-of-care test for any infectious disease target, via modification of the LAMP primer set.","lamp_id":[{"id":"LPB00832","pathogen":"Staphylococcus aureus","target":"mecA"}]},{"id":459,"pmid":35509356,"title":"Fluorescent Detection of Methicillin Resistant Staphylococcus aureus by Loop-mediated Isothermal Amplification Assisted with Streptavidin-coated Quantum Dots","year":2022,"journal":"Avicenna Journal of Medical Biotechnology","authors":"Aily Aliasgharian, Pooria Gill, Mohammad Ahanjan, Adele Rafati","doi":"10.18502\/ajmb.v14i1.8546","country":"","institute":"","deparment":"","abstract":"Background: Methicillin Resistance Staphylococcus aureus (MRSA) could be considered as a major concern in medicine can cause nosocomial infection and bacteremia, especially in patients using catheter and household medical devices.\n\nMethods: Using molecular diagnostic methods are important for identification of MRSA from the Methicillin Sensitive Staphylococcus aureus (MSSA). Here we described a fluorescent assay using biotin-labelling Loop-mediated isothermal amplification (LAMP) method assisted with streptavidin-coated Quantum Dots (QDs) for detection of MRSA. For comparison, another fluorescent assay using LAMP assisted with Green Viewer (GV; a fluorescent dye) was applied for detection of MRSA. The mecA gene was selected as the target for amplification by LAMP and for biotin-labeling of the LAMP amplicons, biotin-11-dUTP was mixed with the dNTPs (deoxy Nucleotide Phosphates) in LAMP reaction. For determining the clinical performance of the developed assay, 30 blood samples with MRSA positive results were tested with QD-LAMP, the conventional LAMP, GV-LAMP, and Polymerase Chain Reaction (PCR).\n\nResults: Obtained results indicated that % sensitivity of QD-LAMP was 86.66% for detection of mecA positive MRSA samples; however, the Limit of Detection (LoD) of QD-LAMP was 1.5\u00d710^4 Colony Forming Unit (CFU).\n\nConclusion: The results suggested that the QD-LAMP assay was easy to operate and could be used for detection of MRSA in parallel to the blood culture with less sensitivity for detection of bacteremia and pediatric septicemia with low counts of MRSA.","lamp_id":[{"id":"LPB00834","pathogen":"Staphylococcus aureus","target":"mecA"}]},{"id":460,"pmid":21399774,"title":"A magnetic bead-based assay for the rapid detection of methicillin-resistant Staphylococcus aureus by using a microfluidic system with integrated loop-mediated isothermal amplification","year":2011,"journal":"Lab on a Chip","authors":"Chih-Hung Wang, Kang-Yi Lien, Jiunn-Jong Wu, Gwo-Bin Lee","doi":"10.1039\/c0lc00430h","country":"","institute":"","deparment":"","abstract":"This study reports a new diagnostic assay for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) by combing nucleic acid extraction and isothermal amplification of target nucleic acids in a magnetic bead-based microfluidic system. By using specific probe-conjugated magnetic beads, the target deoxyribonucleic acid (DNA) of the MRSA can be specifically recognized and hybridized onto the surface of the magnetic beads which are then mixed with clinical sample lysates. This is followed by purifying and concentrating the target DNA from the clinical sample lysates by applying a magnetic field. Nucleic acid amplification of the target genes can then be performed by the use of a loop-mediated isothermal amplification (LAMP) process via the incorporation of a built-in micro temperature control module, followed by analyzing the optical density (OD) of the LAMP amplicons using a spectrophotometer. Significantly, experimental results show that the limit of detection (LOD) for MRSA in the clinical samples is approximately 10 fg \u03bcL(-1) by performing this diagnostic assay in the magnetic bead-based microfluidic system. In addition, the entire diagnostic protocol, from bio-sample pre-treatment to optical detection, can be automatically completed within 60 min. Consequently, this miniature diagnostic assay may become a powerful tool for the rapid purification and detection of MRSA and a potential point-of-care platform for detection of other types of infections.","lamp_id":[{"id":"LPB00835","pathogen":"Staphylococcus aureus","target":"mecA"}]},{"id":461,"pmid":36361893,"title":"Development and Clinical Validation of RT-LAMP-Based Lateral-Flow Devices and Electrochemical Sensor for Detecting Multigene Targets in SARS-CoV-2","year":2022,"journal":"International Journal of Molecular Sciences","authors":"Apoorva Saxena, Pawankumar Rai, Srishti Mehrotra, Samiya Baby, Suman Singh, Vikas Srivastava, Smriti Priya, Sandeep K Sharma","doi":"10.3390\/ijms232113105","country":"","institute":"","deparment":"","abstract":"Consistently emerging variants and the life-threatening consequences of SARS-CoV-2 have prompted worldwide concern about human health, necessitating rapid and accurate point-of-care diagnostics to limit the spread of COVID-19. Still, However, the availability of such diagnostics for COVID-19 remains a major rate-limiting factor in containing the outbreaks. Apart from the conventional reverse transcription polymerase chain reaction, loop-mediated isothermal amplification-based (LAMP) assays have emerged as rapid and efficient systems to detect COVID-19. The present study aims to develop RT-LAMP-based assay system for detecting multiple targets in N, ORF1ab, E, and S genes of the SARS-CoV-2 genome, where the end-products were quantified using spectrophotometry, paper-based lateral-flow devices, and electrochemical sensors. The spectrophotometric method shows a LOD of 10 ag\/\u00b5L for N, ORF1ab, E genes and 100 ag\/\u00b5L for S gene in SARS-CoV-2. The developed lateral-flow devices showed an LOD of 10 ag\/\u00b5L for all four gene targets in SARS-CoV-2. An electrochemical sensor developed for N-gene showed an LOD and E-strip sensitivity of log 1.79 \u00b1 0.427 pg\/\u00b5L and log 0.067 \u00b5A\/pg \u00b5L-1\/mm2, respectively. The developed assay systems were validated with the clinical samples from COVID-19 outbreaks in 2020 and 2021. This multigene target approach can effectively detect emerging COVID-19 variants using combination of various analytical techniques at testing facilities and in point-of-care settings.","lamp_id":[{"id":"LPB00836","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00837","pathogen":"SARS-CoV-2","target":"ORF1ab"},{"id":"LPB00838","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00839","pathogen":"SARS-CoV-2","target":"S"}]},{"id":462,"pmid":null,"title":"Development and Evaluation of RT-LAMP Assays to Identify Variants of SARS-CoV-2","year":2022,"journal":"bioRxiv","authors":"Gun-Soo Park, Seong-Jun Kim, Jin-Soo Maeng","doi":"10.1101\/2022.06.16.496383","country":"","institute":"","deparment":"","abstract":null,"lamp_id":[{"id":"LPB00844","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00845","pathogen":"SARS-CoV-2 (Alpha, Beta, Gamma, Omicron)","target":"ORF1a"},{"id":"LPB00846","pathogen":"SARS-CoV-2 (Delta)","target":"S"},{"id":"LPB00847","pathogen":"SARS-CoV-2","target":"S"},{"id":"LPB00848","pathogen":"SARS-CoV-2 (Omicron)","target":"S"},{"id":"LPB00849","pathogen":"SARS-CoV-2","target":"S"}]},{"id":463,"pmid":35361863,"title":"Improving the specificity of nucleic acid detection with endonuclease-actuated degradation","year":2022,"journal":"Communications Biology","authors":"Roger S. Zou, Momcilo Gavrilov, Yang Liu, Dominique Rasoloson, Madison Conte, Justin Hardick, Leo Shen, Siqi Chen, Andrew Pekosz, Geraldine Seydoux, Yukari C. Manabe, Taekjip Ha","doi":"10.1038\/s42003-022-03242-x","country":"","institute":"","deparment":"","abstract":"Nucleic acid detection is essential for numerous biomedical applications, but often requires complex protocols and\/or suffers false-positive readouts. Here, we describe SENTINEL, an approach that combines isothermal amplification with a sequence-specific degradation method to detect nucleic acids with high sensitivity and sequence-specificity. Target single-stranded RNA or double-stranded DNA molecules are amplified by loop-mediated isothermal amplification (LAMP) and subsequently degraded by the combined action of lambda exonuclease and a sequence-specific DNA endonuclease (e.g., Cas9). By combining the sensitivity of LAMP with the precision of DNA endonucleases, the protocol achieves attomolar limits of detection while differentiating between sequences that differ by only one or two base pairs. The protocol requires less than an hour to complete using a 65\u2009\u00b0C heat block and fluorometer, and detects SARS-CoV-2 virus particles in human saliva and nasopharyngeal swabs with high sensitivity.","lamp_id":[{"id":"LPB00850","pathogen":"SARS-CoV-2","target":"N"}]},{"id":464,"pmid":34188222,"title":"LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding","year":2021,"journal":"Nature Biotechnology","authors":"Kerstin U. Ludwig, Ricarda M. Schmithausen, David Li, Max L. Jacobs, Ronja Hollstein, Katja Blumenstock, Jana Liebing, Miko\u0142aj S\u0142abicki, Amir Ben-Shmuel, Ofir Israeli, Shay Weiss, Thomas S. Ebert, Nir Paran, Wibke R\u00fcdiger, Gero Wilbring, David Feldman, B\u00e4rbel Lippke, Nina Ishorst, Lara M. Hochfeld, Eva C. Beins, Ines H. Kaltheuner, Maximilian Schmitz, Aliona W\u00f6hler, Manuel D\u00f6hla, Esther Sib, Marius Jentzsch, Jacob D. Borrajo, Jonathan Strecker, Julia Reinhardt, Brian Cleary, Matthias Geyer, Michael H\u00f6lzel, Rhiannon Macrae, Markus M. N\u00f6then, Per Hoffmann, Martin Exner, Aviv Regev, Feng Zhang, Jonathan L. Schmid-Burgk","doi":"10.1038\/s41587-021-00966-9","country":"","institute":"","deparment":"","abstract":"Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing COVID-19 outbreaks. Here, we present LAMP-Seq, a modified, highly scalable reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method. Unpurified biosamples are barcoded and amplified in a single heat step, and pooled products are analyzed en masse by sequencing. Using commercial reagents, LAMP-Seq has a limit of detection of ~2.2 molecules\/\u00b5l at 95% confidence, and near-perfect specificity for SARS-CoV-2 given its sequence readout. Clinical validation of an open-source protocol with 676 swab samples, 98 of which were deemed positive by standard RT-qPCR, demonstrated 100% sensitivity in individuals with Ct values up to 33, and a specificity of 99.7%, at material cost of 2.73 USD per sample. With a time-to-result less than 24 hours, low cost, and little new infrastructure requirement, LAMP-Seq can be readily deployed for frequent testing as part of an integrated public health surveillance program.","lamp_id":[{"id":"LPB00851","pathogen":"SARS-CoV-2","target":"N"}]},{"id":465,"pmid":34818890,"title":"Universally Stable and Precise CRISPR-LAMP Detection Platform for Precise Multiple Respiratory Tract Virus Diagnosis Including Mutant SARS-CoV-2 Spike N501Y","year":2021,"journal":"Analytical Chemistry","authors":"Tong Zhang, Wei Zhao, Wang Zhao, Yuying Si, Nianzhen Chen, Xi Chen, Xinlian Zhang, Lieying Fan, Guodong Sui","doi":"10.1021\/acs.analchem.1c04065","country":"","institute":"","deparment":"","abstract":"Nowadays, rapid and accurate diagnosis of respiratory tract viruses is an urgent need to prevent another epidemic outbreak. To overcome this problem, we have developed a clustered, regularly interspaced short palindromic repeats (CRISPR) loop mediated amplification (LAMP) technology to detect influenza A virus, influenza B virus, respiratory syncytial A virus, respiratory syncytial B virus, and severe acute respiratory syndrome coronavirus 2, including variants of concern (B.1.1.7), which utilized CRISPR-associated protein 12a (Cas12a) to advance LAMP technology with the sensitivity increased 10 times. To reduce aerosol contamination in CRISPR-LAMP technology, an uracil-DNA-glycosylase-reverse transcription-LAMP system was also developed which can effectively remove dUTP-incorporated LAMP amplicons. In vitro Cas12a cleavage reaction with 28 crRNAs showed that there were no position constraints for Cas12a\/CRISPR RNA (crRNA) recognition and cleavage in LAMP amplicons, and even the looped position of LAMP amplicons could be effectively recognized and cleaved. Wild-type or spike N501Y can be detected with a limit of detection of 10 copies\/\u03bcL (wild-type) even at a 1% ratio level on the background (spike N501Y). Combining UDG-RT-LAMP technology, CRISPR-LAMP design, and mutation detection design, we developed a CRISPR-LAMP detection platform that can precisely diagnose pathogens with better stability and significantly improved point mutation detection efficiency.","lamp_id":[{"id":"LPB00852","pathogen":"Influenza A virus (H1)","target":"HA"},{"id":"LPB00853","pathogen":"Influenza A virus (H3N2)","target":"HA"},{"id":"LPB00854","pathogen":"RSV A","target":"M"},{"id":"LPB00855","pathogen":"RSV B","target":"G"},{"id":"LPB00856","pathogen":"Influenza B virus","target":"NS1 and NEP"},{"id":"LPB00857","pathogen":"SARS-CoV-2","target":"N"},{"id":"LPB00858","pathogen":"SARS-CoV-2","target":"RdRp"},{"id":"LPB00859","pathogen":"SARS-CoV-2","target":"S"}]},{"id":466,"pmid":36398425,"title":"Multiplexed Ultrasensitive Sample-to-Answer RT-LAMP Chip for the Identification of SARS-CoV-2 and Influenza Viruses","year":2022,"journal":"Advanced Materials","authors":"Minsun Song, SoonGweon Hong, Luke P Lee","doi":"10.1002\/adma.202207138","country":"","institute":"","deparment":"","abstract":"Prompt on-site diagnosis of SARS-CoV-2 with other respiratory infections will have minimized the global impact of the COVID-19 pandemic through rapid, effective management. However, no such multiplex point-of-care (POC) chip has satisfied a suitable sensitivity of gold-standard nucleic acid amplification tests (NAATs). Here, a rapid multiplexed ultrasensitive sample-to-answer loop-mediated isothermal amplification (MUSAL) chip operated by simple LED-driven photothermal amplification to detect six targets from single-swab sampling is presented. First, the MUSAL chip allows ultrafast on-chip sample preparation with \u2248500-fold preconcentration at a rate of 1.2 mL min-1 . Second, the chip enables contamination-free amplification using autonomous target elution into on-chip reagents by photothermal activation. Finally, the chip accomplishes multiplexed on-chip diagnostics of SARS-CoV-2 and influenza viruses with a limit of detection (LoD) of 0.5 copies\/\u00b5L . The rapid, ultrasensitive, cost-effective sample-to-answer chip with a multiplex capability will allow timely management of various pandemics situations that may be faced shortly.","lamp_id":[{"id":"LPB00860","pathogen":"SARS-CoV-2","target":"ORF1a"},{"id":"LPB00861","pathogen":"SARS-CoV-2","target":"E"},{"id":"LPB00862","pathogen":"SARS-CoV-2","target":"N"}]}]}