{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,3]],"date-time":"2026-11-03T14:41:50Z","timestamp":1793716910952,"version":"4.6.1"},"update-to":[{"DOI":"10.1371\/journal.pntd.0010112","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2022,1,19]],"date-time":"2022-01-19T00:00:00Z","timestamp":1642550400000}}],"reference-count":39,"publisher":"Public Library of Science (PLoS)","issue":"1","license":[{"start":{"date-parts":[[2022,1,6]],"date-time":"2022-01-06T00:00:00Z","timestamp":1641427200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002873","name":"chulalongkorn university","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100002873","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosntds.org"],"crossmark-restriction":false},"short-container-title":["PLoS Negl Trop Dis"],"abstract":"<jats:sec id=\"sec001\">\n<jats:title>Background<\/jats:title>\n<jats:p>One of the key barriers preventing rapid diagnosis of leptospirosis is the lack of available sensitive point-of-care testing. This study aimed to develop and validate a clustered regularly-interspaced short palindromic repeat (CRISPR)\/CRISPR-associated protein 12a (CRISPR\/Cas12a) platform combined with isothermal amplification to detect leptospires from extracted patient DNA samples.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methodology\/Principal findings<\/jats:title>\n<jats:p>A Recombinase Polymerase Amplification (RPA)-CRISPR\/Cas12a-fluorescence assay was designed to detect the <jats:italic>lipL32<\/jats:italic> gene of pathogenic <jats:italic>Leptospira<\/jats:italic> spp. The assays demonstrated a limit of detection (LOD) of 100 cells\/mL, with no cross-reactivity against several other acute febrile illnesses. The clinical performance of the assay was validated with DNA extracted from 110 clinical specimens and then compared to results from qPCR detection of <jats:italic>Leptospira<\/jats:italic> spp. The RPA-CRISPR\/Cas12a assay showed 85.2% sensitivity, 100% specificity, and 92.7% accuracy. The sensitivity increased on days 4\u20136 after the fever onset and decreased after day 7. The specificity was consistent for several days after the onset of fever. The overall performance of the RPA-CRISPR\/Cas12a platform was better than the commercial rapid diagnostic test (RDT). We also developed a lateral flow detection assay (LFDA) combined with RPA-CRISPR\/Cas12a to make the test more accessible and easier to interpret. The combined LFDA showed a similar LOD of 100 cells\/mL and could correctly distinguish between known positive and negative clinical samples in a pilot study.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Conclusions\/Significance<\/jats:title>\n<jats:p>The RPA-CRISPR\/Cas12 targeting the <jats:italic>lipL32<\/jats:italic> gene demonstrated acceptable sensitivity and excellent specificity for detection of leptospires. This assay might be an appropriate test for acute leptospirosis screening in limited-resource settings.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pntd.0010112","type":"journal-article","created":{"date-parts":[[2022,1,6]],"date-time":"2022-01-06T18:33:46Z","timestamp":1641494026000},"page":"e0010112","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":59,"title":["Rapid and sensitive point-of-care detection of Leptospira by RPA-CRISPR\/Cas12a targeting lipL32"],"prefix":"10.1371","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4468-4750","authenticated-orcid":true,"given":"Sirawit","family":"Jirawannaporn","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Umaporn","family":"Limothai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sasipha","family":"Tachaboon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Janejira","family":"Dinhuzen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patcharakorn","family":"Kiatamornrak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Watchadaporn","family":"Chaisuriyong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9504-3614","authenticated-orcid":true,"given":"Jom","family":"Bhumitrakul","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5972-6648","authenticated-orcid":true,"given":"Oraphan","family":"Mayuramart","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2668-110X","authenticated-orcid":true,"given":"Sunchai","family":"Payungporn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8544-8132","authenticated-orcid":true,"given":"Nattachai","family":"Srisawat","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,1,6]]},"reference":[{"issue":"9","key":"pntd.0010112.ref001","doi-asserted-by":"crossref","first-page":"e0003898","DOI":"10.1371\/journal.pntd.0003898","article-title":"Global Morbidity and Mortality of Leptospirosis: A Systematic Review.","volume":"9","author":"F Costa","year":"2015","journal-title":"PLoS Neglected Tropical Diseases"},{"issue":"2","key":"pntd.0010112.ref002","doi-asserted-by":"crossref","first-page":"296","DOI":"10.1128\/CMR.14.2.296-326.2001","article-title":"Leptospirosis","volume":"14","author":"PN Levett","year":"2001","journal-title":"Clinical Microbiology Reviews"},{"key":"pntd.0010112.ref003","first-page":"105183","article-title":"Leptospirosis: Increasing importance in developing countries","volume":"201","author":"ZMP Soo","year":"2019","journal-title":"Acta Tropica"},{"key":"pntd.0010112.ref004","volume-title":"Human leptospirosis: guidance for diagnosis, surveillance and control","author":"World Health O","year":"2003"},{"issue":"1","key":"pntd.0010112.ref005","first-page":"199","article-title":"Leptospirosis Diagnosis: Competancy of Various Laboratory Tests","volume":"8","author":"SV Budihal","year":"2014","journal-title":"Journal of Clinical and Diagnostic Research"},{"issue":"3","key":"pntd.0010112.ref006","first-page":"195","article-title":"Laboratory diagnosis of leptospirosis","volume":"51","author":"SN Ahmad","year":"2005","journal-title":"Journal of Postgraduate Medicine"},{"issue":"1","key":"pntd.0010112.ref007","doi-asserted-by":"crossref","first-page":"3215","DOI":"10.1038\/s41598-021-82290-5","article-title":"Human, animal, water source interactions and leptospirosis in Thailand.","volume":"11","author":"U Narkkul","year":"2021","journal-title":"Scientific Reports"},{"issue":"5","key":"pntd.0010112.ref008","doi-asserted-by":"crossref","first-page":"4953","DOI":"10.3390\/ijerph110504953","article-title":"Development of a recombinase polymerase amplification assay for the detection of pathogenic Leptospira","volume":"11","author":"A Ahmed","year":"2014","journal-title":"International Journal of Environmental Research and Public Health"},{"issue":"2","key":"pntd.0010112.ref009","doi-asserted-by":"crossref","first-page":"e0009159","DOI":"10.1371\/journal.pntd.0009159","article-title":"A prospective study to evaluate the accuracy of rapid diagnostic tests for diagnosis of human leptospirosis: Result from THAI-LEPTO AKI study.","volume":"15","author":"J Dinhuzen","year":"2021","journal-title":"PLoS Neglected Tropical Diseases"},{"issue":"4","key":"pntd.0010112.ref010","doi-asserted-by":"crossref","first-page":"614","DOI":"10.4269\/ajtmh.2011.10-0473","article-title":"Accuracy of loop-mediated isothermal amplification for diagnosis of human leptospirosis in Thailand","volume":"84","author":"P Sonthayanon","year":"2011","journal-title":"American Journal of Tropical Medicine and Hygiene"},{"issue":"7","key":"pntd.0010112.ref011","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1373\/clinchem.2015.245829","article-title":"Recombinase Polymerase Amplification for Diagnostic Applications","volume":"62","author":"RK Daher","year":"2016","journal-title":"Clinical Chemistry"},{"issue":"6336","key":"pntd.0010112.ref012","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1126\/science.aam9321","article-title":"Nucleic acid detection with CRISPR-Cas13a\/C2c2","volume":"356","author":"JS Gootenberg","year":"2017","journal-title":"Science"},{"key":"pntd.0010112.ref013","article-title":"Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6","author":"JS Gootenberg","year":"2018","journal-title":"Science"},{"key":"pntd.0010112.ref014","article-title":"CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity","author":"JS Chen","year":"2018","journal-title":"Science"},{"issue":"4","key":"pntd.0010112.ref015","first-page":"400","article-title":"Leptospirosis-associated acute kidney injury.","volume":"32","author":"F Daher Ede","year":"2010","journal-title":"J Bras Nefrol"},{"key":"pntd.0010112.ref016","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.vetimm.2015.12.004","article-title":"Leptospira spp.: Novel insights into host-pathogen interactions","volume":"176","author":"LG Fernandes","year":"2016","journal-title":"Veterinary Immunology and Immunopathology"},{"issue":"4","key":"pntd.0010112.ref017","doi-asserted-by":"crossref","first-page":"2276","DOI":"10.1128\/IAI.68.4.2276-2285.2000","article-title":"The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection","volume":"68","author":"DA Haake","year":"2000","journal-title":"Infection and Immunity"},{"issue":"1","key":"pntd.0010112.ref018","doi-asserted-by":"crossref","first-page":"e2672","DOI":"10.1371\/journal.pntd.0002672","article-title":"Leptospira interrogans Binds to Cadherins.","volume":"8","author":"K Evangelista","year":"2014","journal-title":"PLoS Neglected Tropical Diseases"},{"issue":"1","key":"pntd.0010112.ref019","doi-asserted-by":"crossref","first-page":"5","DOI":"10.4067\/S0716-97602009000100001","article-title":"Leptospirosis research: fast, easy and reliable enumeration of mobile leptospires.","volume":"42","author":"S Schreier","year":"2009","journal-title":"Biological Research"},{"issue":"3","key":"pntd.0010112.ref020","doi-asserted-by":"crossref","first-page":"e0006319","DOI":"10.1371\/journal.pntd.0006319","article-title":"Thai-Lepto-on-admission probability (THAI-LEPTO) score as an early tool for initial diagnosis of leptospirosis: Result from Thai-Lepto AKI study group.","volume":"12","author":"T Sukmark","year":"2018","journal-title":"PLoS Neglected Tropical Diseases"},{"issue":"1","key":"pntd.0010112.ref021","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1016\/j.mimet.2012.06.009","article-title":"Novel TaqMan(R) PCR for detection of Leptospira species in urine and blood: pit-falls of in silico validation.","volume":"91","author":"S Villumsen","year":"2012","journal-title":"Journal of Microbiological Methods"},{"issue":"12","key":"pntd.0010112.ref022","doi-asserted-by":"crossref","first-page":"e0143367","DOI":"10.1371\/journal.pone.0143367","article-title":"Neutrophil Gelatinase Associated Lipocalin (NGAL) in Leptospirosis Acute Kidney Injury: A Multicenter Study in Thailand.","volume":"10","author":"N Srisawat","year":"2015","journal-title":"PloS One"},{"issue":"7","key":"pntd.0010112.ref023","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1038\/s41587-020-0513-4","article-title":"CRISPR\u2013Cas12-based detection of SARS-CoV-2","volume":"38","author":"JP Broughton","year":"2020","journal-title":"Nature Biotechnology"},{"issue":"4","key":"pntd.0010112.ref024","doi-asserted-by":"crossref","first-page":"400","DOI":"10.1177\/1535370220963793","article-title":"Detection of severe acute respiratory syndrome coronavirus 2 and influenza viruses based on CRISPR-Cas12a","volume":"246","author":"O Mayuramart","year":"2021","journal-title":"Experimental Biology and Medicine"},{"issue":"1","key":"pntd.0010112.ref025","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1186\/1471-2334-2-13","article-title":"A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp.","volume":"2","author":"LD Smythe","year":"2002","journal-title":"BMC Infectious Diseases"},{"key":"pntd.0010112.ref026","doi-asserted-by":"crossref","first-page":"114092","DOI":"10.1016\/j.jviromet.2021.114092","article-title":"Comparative performance of CRISPR-Cas12a assays for SARS-CoV-2 detection tested with RNA extracted from clinical specimens","volume":"290","author":"P Nimsamer","year":"2021","journal-title":"Journal of Virological Methods"},{"issue":"17","key":"pntd.0010112.ref027","doi-asserted-by":"crossref","first-page":"1685","DOI":"10.1056\/NEJMcibr1704902","article-title":"A CRISPR Way to Diagnose Infectious Diseases.","volume":"377","author":"AM Caliendo","year":"2017","journal-title":"New England Journal of Medicine"},{"issue":"7","key":"pntd.0010112.ref028","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1016\/j.tibtech.2018.12.005","article-title":"CRISPR\/Cas Systems towards Next-Generation Biosensing","volume":"37","author":"Y Li","year":"2019","journal-title":"Trends in Biotechnology"},{"issue":"10","key":"pntd.0010112.ref029","doi-asserted-by":"crossref","first-page":"2986","DOI":"10.1038\/s41596-019-0210-2","article-title":"SHERLOCK: nucleic acid detection with CRISPR nucleases.","volume":"14","author":"MJ Kellner","year":"2019","journal-title":"Nature Protocols"},{"issue":"6","key":"pntd.0010112.ref030","doi-asserted-by":"crossref","first-page":"e0129682","DOI":"10.1371\/journal.pone.0129682","article-title":"Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection.","volume":"10","author":"A Abd El Wahed","year":"2015","journal-title":"PLOS ONE"},{"issue":"2","key":"pntd.0010112.ref031","doi-asserted-by":"crossref","first-page":"e1607","DOI":"10.1371\/journal.pone.0001607","article-title":"Genome sequence of the saprophyte Leptospira biflexa provides insights into the evolution of Leptospira and the pathogenesis of leptospirosis","volume":"3","author":"M Picardeau","year":"2008","journal-title":"PloS One"},{"issue":"3","key":"pntd.0010112.ref032","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1016\/j.diagmicrobio.2015.03.024","article-title":"Optimization of LipL32 PCR assay for increased sensitivity in diagnosing leptospirosis","volume":"82","author":"RL Galloway","year":"2015","journal-title":"Diagnostic Microbiology and Infectious Disease"},{"key":"pntd.0010112.ref033","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1186\/1475-2875-13-99","article-title":"Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis","volume":"13","author":"S Kersting","year":"2014","journal-title":"Malaria Journal"},{"issue":"3","key":"pntd.0010112.ref034","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1016\/j.diagmicrobio.2009.03.014","article-title":"Detection of pathogenic Leptospira spp. through TaqMan polymerase chain reaction targeting the LipL32 gene","volume":"64","author":"RA Stoddard","year":"2009","journal-title":"Diagn Microbiol Infect Dis"},{"issue":"1","key":"pntd.0010112.ref035","doi-asserted-by":"crossref","first-page":"e1991","DOI":"10.1371\/journal.pntd.0001991","article-title":"Risk factors and predictors of severe leptospirosis in New Caledonia.","volume":"7","author":"S Tubiana","year":"2013","journal-title":"PLoS Negl Trop Dis"},{"issue":"1","key":"pntd.0010112.ref036","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.medmal.2012.11.005","article-title":"Diagnosis and epidemiology of leptospirosis.","volume":"43","author":"M Picardeau","year":"2013","journal-title":"M\u00e9decine et Maladies Infectieuses"},{"issue":"17","key":"pntd.0010112.ref037","doi-asserted-by":"crossref","first-page":"5538","DOI":"10.1074\/jbc.RA120.012933","article-title":"CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects","volume":"295","author":"K Murugan","year":"2020","journal-title":"J Biol Chem"},{"key":"pntd.0010112.ref038","first-page":"11","article-title":"Seroepidemiology of leptospirosis in dogs from rural and slum communities of Los Rios Region, Chile.","author":"M Lelu","year":"2015","journal-title":"BMC veterinary research"},{"issue":"1","key":"pntd.0010112.ref039","doi-asserted-by":"crossref","first-page":"e16236","DOI":"10.1371\/journal.pone.0016236","article-title":"Diagnostic Accuracy of Real-Time PCR Assays Targeting 16S rRNA and lipl32 Genes for Human Leptospirosis in Thailand: A Case-Control Study.","volume":"6","author":"J Thaipadunpanit","year":"2011","journal-title":"PloS One"}],"updated-by":[{"DOI":"10.1371\/journal.pntd.0010112","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2022,1,19]],"date-time":"2022-01-19T00:00:00Z","timestamp":1642550400000}}],"container-title":["PLOS Neglected Tropical Diseases"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pntd.0010112","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,1,19]],"date-time":"2022-01-19T18:37:22Z","timestamp":1642617442000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pntd.0010112"}},"subtitle":[],"editor":[{"given":"Kelly","family":"Hawley","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2022,1,6]]},"references-count":39,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2022,1,6]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pntd.0010112","relation":{"new_version":[{"id-type":"doi","id":"10.1371\/journal.pntd.0010112","asserted-by":"object"}]},"ISSN":["1935-2735"],"issn-type":[{"value":"1935-2735","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,1,6]]}}}