{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T22:30:03Z","timestamp":1792794603972,"version":"4.5.0"},"reference-count":46,"publisher":"Oxford University Press (OUP)","issue":"7","license":[{"start":{"date-parts":[[2020,2,27]],"date-time":"2020-02-27T00:00:00Z","timestamp":1582761600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000774","name":"Defense Threat Reduction Agency","doi-asserted-by":"publisher","award":["MCDC-18-01-01-007"],"award-info":[{"award-number":["MCDC-18-01-01-007"]}],"id":[{"id":"10.13039\/100000774","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000774","name":"Defense Threat Reduction Agency","doi-asserted-by":"publisher","award":["W15QKN-16-9-1002"],"award-info":[{"award-number":["W15QKN-16-9-1002"]}],"id":[{"id":"10.13039\/100000774","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000861","name":"Burroughs Wellcome Fund","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000861","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["T32GM112592"],"award-info":[{"award-number":["T32GM112592"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["DGE-1144469"],"award-info":[{"award-number":["DGE-1144469"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Joseph J. Jacobs Institute for Molecular Engineering for Medicine","id":[{"id":"https:\/\/ror.org\/04c00sd86","id-type":"ROR","asserted-by":"crossref"}]},{"DOI":"10.13039\/100006961","name":"Caltech","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100006961","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000774","name":"DTRA","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000774","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2020,4,17]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.<\/jats:p>","DOI":"10.1093\/nar\/gkaa099","type":"journal-article","created":{"date-parts":[[2020,2,6]],"date-time":"2020-02-06T23:23:18Z","timestamp":1581031398000},"page":"e42-e42","source":"Crossref","is-referenced-by-count":219,"title":["Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification"],"prefix":"10.1093","volume":"48","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8948-319X","authenticated-orcid":false,"given":"Justin C","family":"Rolando","sequence":"first","affiliation":[{"name":"Division of Chemistry and Chemical Engineering,\u00a0California Institute of Technology 1200 E.\u00a0California Boulevard, Pasadena, CA 91125,\u00a0USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7585-3794","authenticated-orcid":false,"given":"Erik","family":"Jue","sequence":"first","affiliation":[{"name":"Division of Biology and Biological Engineering, California Institute of Technology 1200 E.\u00a0California Boulevard, Pasadena, CA\u00a091125, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1842-4835","authenticated-orcid":false,"given":"Jacob T","family":"Barlow","sequence":"first","affiliation":[{"name":"Division of Biology and Biological Engineering, California Institute of Technology 1200 E.\u00a0California Boulevard, Pasadena, CA\u00a091125, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3680-4399","authenticated-orcid":false,"given":"Rustem F","family":"Ismagilov","sequence":"first","affiliation":[{"name":"Division of Chemistry and Chemical Engineering,\u00a0California Institute of Technology 1200 E.\u00a0California Boulevard, Pasadena, CA 91125,\u00a0USA"},{"name":"Division of Biology and Biological Engineering, California Institute of Technology 1200 E.\u00a0California Boulevard, Pasadena, CA\u00a091125, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2020,2,27]]},"reference":[{"key":"2020040912554222300_B1","doi-asserted-by":"crossref","first-page":"2934","DOI":"10.1021\/acsnano.6b08274","article-title":"Highly stable and sensitive nucleic acid amplification and cell-phone-based readout","volume":"11","author":"Kong","year":"2017","journal-title":"ACS Nano"},{"key":"2020040912554222300_B2","doi-asserted-by":"crossref","first-page":"6580","DOI":"10.1021\/acs.analchem.8b00269","article-title":"Strand displacement probes combined with isothermal nucleic acid amplification for instrument-free detection from complex samples","volume":"90","author":"Phillips","year":"2018","journal-title":"Anal. Chem."},{"key":"2020040912554222300_B3","doi-asserted-by":"crossref","first-page":"e63","DOI":"10.1093\/nar\/28.12.e63","article-title":"Loop-mediated isothermal amplification of DNA","volume":"28","author":"Notomi","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"2020040912554222300_B4","doi-asserted-by":"crossref","first-page":"e0006900","DOI":"10.1371\/journal.pntd.0006900","article-title":"Rapid and sensitive detection of Chlamydia trachomatis sexually transmitted infections in resource-constrained settings in Thailand at the point-of-care","volume":"12","author":"Somboonna","year":"2018","journal-title":"PLoS Negl. Trop. Dis."},{"key":"2020040912554222300_B5","doi-asserted-by":"crossref","first-page":"183167","DOI":"10.1155\/2014\/183167","article-title":"Chlamydia trachomatis and genital mycoplasmas: pathogens with an impact on human reproductive health","volume":"2014","author":"Ljubin-Sternak","year":"2014","journal-title":"J. Pathog."},{"key":"2020040912554222300_B6","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1016\/j.tjog.2011.07.001","article-title":"Diagnosis of Chlamydia infection in women","volume":"50","author":"Su","year":"2011","journal-title":"Taiwan. J. Obstet. Gynecol."},{"key":"2020040912554222300_B7","article-title":"Supplemental Tables for Interference Testing in Clinical Chemistry, 1st Edition, EP 37","author":"CLSI","year":"2018"},{"key":"2020040912554222300_B8","volume-title":"Interference Testing in Clinical Chemistry, EP07-A2","author":"CLSI","year":"2005"},{"key":"2020040912554222300_B9","doi-asserted-by":"crossref","first-page":"2706","DOI":"10.1002\/elps.201900167","article-title":"Mathematical model to reduce loop mediated isothermal amplification (LAMP) false-positive diagnosis","volume":"40","author":"Schneider","year":"2019","journal-title":"Electrophoresis"},{"key":"2020040912554222300_B10","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1016\/j.jviromet.2011.01.009","article-title":"Real-time target-specific detection of loop-mediated isothermal amplification for white spot syndrome virus using fluorescence energy transfer-based probes","volume":"173","author":"Chou","year":"2011","journal-title":"J. Virol. Methods"},{"key":"2020040912554222300_B11","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-018-23930-1","article-title":"Real-time detection and monitoring of loop mediated amplification (LAMP) reaction using Self-quenching and De-quenching fluorogenic probes","volume":"8","author":"Gadkar","year":"2018","journal-title":"Sci. Rep."},{"key":"2020040912554222300_B12","doi-asserted-by":"crossref","first-page":"3562","DOI":"10.1021\/acs.analchem.5b04054","article-title":"Quenching of unincorporated amplification signal reporters in reverse-transcription loop-mediated isothermal amplification enabling bright, single-step, closed-tube, and multiplexed detection of RNA viruses","volume":"88","author":"Ball","year":"2016","journal-title":"Anal. 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Cell Probes"},{"key":"2020040912554222300_B15","doi-asserted-by":"crossref","first-page":"81","DOI":"10.13031\/2013.38509","article-title":"FRET-based assimilating probe for sequence-specific real-time monitoring of loop-mediated isothermal amplification (LAMP)","volume":"4","author":"Kubota","year":"2011","journal-title":"Biol. Eng. Trans."},{"key":"2020040912554222300_B16","doi-asserted-by":"crossref","first-page":"3314","DOI":"10.1021\/ac504387c","article-title":"Robust strand exchange reactions for the sequence-specific, real-time detection of nucleic acid amplicons","volume":"87","author":"Jiang","year":"2015","journal-title":"Anal. Chem."},{"key":"2020040912554222300_B17","first-page":"1","article-title":"Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay","volume":"7","author":"Liu","year":"2017","journal-title":"Sci. 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Chem."},{"key":"2020040912554222300_B23","doi-asserted-by":"crossref","first-page":"1034","DOI":"10.1021\/acs.analchem.8b04324","article-title":"Real-time, digital lamp with commercial microfluidic chips reveals the interplay of efficiency, speed, and background amplification as a function of reaction temperature and time","volume":"91","author":"Rolando","year":"2019","journal-title":"Anal. Chem."},{"key":"2020040912554222300_B24","doi-asserted-by":"crossref","first-page":"e0163060","DOI":"10.1371\/journal.pone.0163060","article-title":"Instrument for real-time digital nucleic acid amplification on custom microfluidic devices","volume":"11","author":"Selck","year":"2016","journal-title":"PLoS One"},{"key":"2020040912554222300_B25","doi-asserted-by":"crossref","first-page":"e147","DOI":"10.1371\/journal.pntd.0000147","article-title":"Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense","volume":"2","author":"Njiru","year":"2008","journal-title":"PLoS Negl. Trop. Dis."},{"key":"2020040912554222300_B26","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.mimet.2016.10.020","article-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","volume":"132","author":"Tone","year":"2017","journal-title":"J. Microbiol. Methods"},{"key":"2020040912554222300_B27","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1099\/jmm.0.47216-0","article-title":"Development of a loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia","volume":"57","author":"Uemura","year":"2008","journal-title":"J. Med. Microbiol."},{"key":"2020040912554222300_B28","first-page":"1","article-title":"Development of a multiplex loop-mediated isothermal amplification method for the simultaneous detection of Salmonella spp. and Vibrio parahaemolyticus","volume":"7","author":"Liu","year":"2017","journal-title":"Sci. Rep."},{"key":"2020040912554222300_B29","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-017-04084-y","article-title":"Novel loop-mediated isothermal amplification (LAMP) assay with a universal QProbe can detect SNPs determining races in plant pathogenic fungi","volume":"7","author":"Ayukawa","year":"2017","journal-title":"Sci. Rep."},{"key":"2020040912554222300_B30","doi-asserted-by":"crossref","first-page":"852","DOI":"10.2144\/01304rr03","article-title":"Isothermal amplification and multimerization of DNA by Bst DNA polymerase","volume":"30","author":"Hafner","year":"2001","journal-title":"Biotechniques"},{"key":"2020040912554222300_B31","first-page":"1","article-title":"Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities","volume":"7","author":"Wang","year":"2017","journal-title":"Sci. Rep."},{"key":"2020040912554222300_B32","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1515\/BC.2007.043","article-title":"N.BspD6I DNA nickase strongly stimulates template-independent synthesis of non-palindromic repetitive DNA by Bst DNA polymerase","volume":"388","author":"Zyrina","year":"2007","journal-title":"Biol. 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Lett."},{"key":"2020040912554222300_B36","doi-asserted-by":"crossref","first-page":"1122","DOI":"10.2144\/01315rr01","article-title":"Interrogation of multimeric DNA amplification products by competitive primer extension using Bst DNA polymerase (Large Fragment)","volume":"31","author":"Voisey","year":"2001","journal-title":"Biotechniques"},{"key":"2020040912554222300_B37","doi-asserted-by":"crossref","first-page":"1924","DOI":"10.1039\/C7AN01897E","article-title":"Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA","volume":"143","author":"Meagher","year":"2018","journal-title":"Analyst"},{"key":"2020040912554222300_B38","doi-asserted-by":"crossref","first-page":"e10","DOI":"10.1093\/nar\/gkv877","article-title":"Lack of correlation between reaction speed and analytical sensitivity in isothermal amplification reveals the value of digital methods for optimization: validation using digital real-time RT-LAMP","volume":"44","author":"Khorosheva","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"2020040912554222300_B39","doi-asserted-by":"crossref","first-page":"15.14.11","DOI":"10.1002\/0471142727.mb1514s105","article-title":"Loop-mediated isothermal amplification for detection of nucleic acids","volume":"105","author":"Tanner","year":"2014","journal-title":"Curr. Protoc. Mol. Biol."},{"key":"2020040912554222300_B40","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1038\/nbt.1641","article-title":"Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations","volume":"28","author":"Rissin","year":"2010","journal-title":"Nat. Biotechnol."},{"key":"2020040912554222300_B41","doi-asserted-by":"crossref","first-page":"520","DOI":"10.1021\/nl060227d","article-title":"Digital concentration readout of single enzyme molecules using femtoliter arrays and poisson statistics","volume":"6","author":"Rissin","year":"2006","journal-title":"Nano Lett."},{"key":"2020040912554222300_B42","doi-asserted-by":"crossref","first-page":"11529","DOI":"10.1021\/acs.analchem.7b02809","article-title":"Integrated bacterial identification and antimicrobial susceptibility testing using PCR and high-resolution melt","volume":"89","author":"Athamanolap","year":"2017","journal-title":"Anal. 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