{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T16:00:54Z","timestamp":1792166454156,"version":"4.4.0"},"update-to":[{"DOI":"10.1371\/journal.pntd.0012167","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2024,5,15]],"date-time":"2024-05-15T00:00:00Z","timestamp":1715731200000}}],"reference-count":34,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2024,5,3]],"date-time":"2024-05-03T00:00:00Z","timestamp":1714694400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program","doi-asserted-by":"crossref","award":["2022YFC2604200"],"award-info":[{"award-number":["2022YFC2604200"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100015976","name":"State Key Laboratory of Pathogen and Biosecurity","doi-asserted-by":"crossref","award":["SKLPBS2112"],"award-info":[{"award-number":["SKLPBS2112"]}],"id":[{"id":"10.13039\/501100015976","id-type":"DOI","asserted-by":"crossref"}]}],"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>Plague, caused by the bacterium <jats:italic>Yersinia pestis<\/jats:italic>, is a zoonotic disease that poses considerable threats to human health. Nucleic acid tests are crucial for plague surveillance and the rapid detection of <jats:italic>Y<\/jats:italic>. <jats:italic>pestis<\/jats:italic>. However, inhibitors in complex samples such as soil and animal tissues often hamper nucleic acid detection, leading to a reduced rate of identifying low concentrations of <jats:italic>Y<\/jats:italic>. <jats:italic>pestis<\/jats:italic>. To address this challenge, we developed a sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay for detecting <jats:italic>Y<\/jats:italic>. <jats:italic>pestis<\/jats:italic> DNA from soil and animal tissue samples.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methods<\/jats:title>\n<jats:p>Three genes (<jats:italic>ypo2088<\/jats:italic>, <jats:italic>caf1<\/jats:italic>, and <jats:italic>pla<\/jats:italic>) from <jats:italic>Y<\/jats:italic>. <jats:italic>pestis<\/jats:italic> were used to develop a multi-target ddPCR assay. The limits of detection (LoD), reproducibility, and specificity were assessed for bacterial genomic DNA samples. The ability of the assay to detect low concentrations of <jats:italic>Y<\/jats:italic>. <jats:italic>pestis<\/jats:italic> DNA from simulated soil and mouse liver tissue samples was respectively evaluated and compared with that of quantitative real-time PCR (qPCR).<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Results<\/jats:title>\n<jats:p>The results showed that the ddPCR LoDs ranged from 6.2 to 15.4 copies\/reaction for the target genes, with good reproducibility and high specificity for <jats:italic>Y<\/jats:italic>. <jats:italic>pestis<\/jats:italic>. By testing 130 soil and mouse liver tissue samples spiked with <jats:italic>Y<\/jats:italic>. <jats:italic>pestis<\/jats:italic>, the ddPCR assay exhibited a better sensitivity than that of the qPCR assay used in the study, with LoDs of 10<jats:sup>2<\/jats:sup> colony forming units (CFU)\/100 mg soil and 10<jats:sup>3<\/jats:sup> CFU\/20 mg liver. Moreover, the assay presented good quantitative linearity (R<jats:sup>2<\/jats:sup> = 0.99) for <jats:italic>Y<\/jats:italic>. <jats:italic>pestis<\/jats:italic> at 10<jats:sup>3<\/jats:sup>\u201310<jats:sup>6<\/jats:sup> CFU\/sample for soil and liver samples.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec004\">\n<jats:title>Conclusion<\/jats:title>\n<jats:p>The ddPCR assay presented good performance for detecting <jats:italic>Y<\/jats:italic>. <jats:italic>pestis<\/jats:italic> DNA from soil and mouse tissue samples, showing great potential for improving the detection rate of low concentrations of <jats:italic>Y<\/jats:italic>. <jats:italic>pestis<\/jats:italic> in plague surveillance and facilitating the early diagnosis of plague cases.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pntd.0012167","type":"journal-article","created":{"date-parts":[[2024,5,3]],"date-time":"2024-05-03T17:29:27Z","timestamp":1714757367000},"page":"e0012167","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":14,"title":["Development and evaluation of a multi-target droplet digital PCR assay for highly sensitive and specific detection of Yersinia pestis"],"prefix":"10.1371","volume":"18","author":[{"given":"Yanting","family":"Zhao","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziheng","family":"Yan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kai","family":"Song","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanbing","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leiming","family":"Shen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yiming","family":"Cui","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zongmin","family":"Du","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruifu","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yajun","family":"Song","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lan","family":"Jing","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1648-4823","authenticated-orcid":true,"given":"Yong","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2024,5,3]]},"reference":[{"issue":"1","key":"pntd.0012167.ref001","doi-asserted-by":"crossref","DOI":"10.1128\/JCM.01519-17","article-title":"Plague: recognition, treatment, and prevention","volume":"56","author":"R. Yang","year":"2018","journal-title":"J Clin Microbiol"},{"issue":"1","key":"pntd.0012167.ref002","article-title":"Yersinia pestis and plague: some knowns and unknowns.","volume":"3","author":"R Yang","year":"2023","journal-title":"Zoonoses (Burlingt)."},{"issue":"1","key":"pntd.0012167.ref003","doi-asserted-by":"crossref","DOI":"10.1128\/CMR.00044-19","article-title":"Yersinia pestis: the natural history of plague.","volume":"34","author":"R Barbieri","year":"2020","journal-title":"Clin Microbiol Rev"},{"issue":"5","key":"pntd.0012167.ref004","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1016\/S1473-3099(18)30730-8","article-title":"Epidemiological characteristics of an urban plague epidemic in Madagascar, August\u2013November, 2017: an outbreak report","volume":"19","author":"R Randremanana","year":"2019","journal-title":"Lancet Infect Dis"},{"issue":"2","key":"pntd.0012167.ref005","doi-asserted-by":"crossref","DOI":"10.3201\/eid3002.230759","article-title":"Multiple introductions of Yersinia pestis during urban pneumonic plague epidemic, Madagascar, 2017","volume":"30","author":"V Andrianaivoarimanana","year":"2024","journal-title":"Emerg Infect Dis"},{"key":"pntd.0012167.ref006","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.epidem.2018.05.001","article-title":"The 2017 plague outbreak in Madagascar: data descriptions and epidemic modelling.","volume":"25","author":"VK Nguyen","year":"2018","journal-title":"Epidemics"},{"issue":"8","key":"pntd.0012167.ref007","doi-asserted-by":"crossref","first-page":"e0008251","DOI":"10.1371\/journal.pntd.0008251","article-title":"Human plague: an old scourge that needs new answers.","volume":"14","author":"X Vall\u00e8s","year":"2020","journal-title":"PLoS Negl Trop Dis"},{"issue":"2","key":"pntd.0012167.ref008","doi-asserted-by":"crossref","DOI":"10.3892\/etm.2022.11438","article-title":"Applications of polymerase chain reaction-based methods for the diagnosis of plague (Review).","volume":"24","author":"Y Zhang","year":"2022","journal-title":"Exp Ther Med."},{"issue":"5","key":"pntd.0012167.ref009","doi-asserted-by":"crossref","DOI":"10.1128\/JCM.00036-19","article-title":"Multiplex detection of three select agents directly from blood by use of the genexpert system","volume":"57","author":"PP Banada","year":"2019","journal-title":"J Clin Microbiol"},{"issue":"4","key":"pntd.0012167.ref010","doi-asserted-by":"crossref","first-page":"992","DOI":"10.1111\/jam.12107","article-title":"Evaluation of the FilmArray system for detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis","volume":"114","author":"DR Seiner","year":"2013","journal-title":"J Appl Microbiol"},{"issue":"11","key":"pntd.0012167.ref011","doi-asserted-by":"crossref","first-page":"1546","DOI":"10.1099\/jmm.0.049007-0","article-title":"Evaluation of two multiplex real-time PCR screening capabilities for the detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis in blood samples generated from murine infection models","volume":"61","author":"SA Weller","year":"2012","journal-title":"J Med Microbiol"},{"issue":"1\u20132","key":"pntd.0012167.ref012","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1111\/j.1574-6968.1990.tb13833.x","article-title":"Plasmid content in Yersinia pestis strains of different origin","volume":"67","author":"AA Filippov","year":"1990","journal-title":"FEMS Microbiol Lett"},{"issue":"3","key":"pntd.0012167.ref013","doi-asserted-by":"crossref","first-page":"e32911","DOI":"10.1371\/journal.pone.0032911","article-title":"Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain java 9 mediates high levels of arsenic resistance.","volume":"7","author":"M Eppinger","year":"2012","journal-title":"PLoS One."},{"issue":"8","key":"pntd.0012167.ref014","doi-asserted-by":"crossref","first-page":"3759","DOI":"10.1128\/AEM.67.8.3759-3762.2001","article-title":"Identification of nucleotide sequences for the specific and rapid detection of Yersinia pestis","volume":"67","author":"L Radnedge","year":"2001","journal-title":"Appl Environ Microbiol"},{"issue":"9","key":"pntd.0012167.ref015","doi-asserted-by":"crossref","first-page":"3930","DOI":"10.1128\/IAI.00281-10","article-title":"Delineation and analysis of chromosomal regions specifying Yersinia pestis","volume":"78","author":"A Derbise","year":"2010","journal-title":"Infect Immun"},{"issue":"24","key":"pntd.0012167.ref016","first-page":"238","article-title":"Revision of the international definition of plague cases.","volume":"96","author":"World Health Organization","year":"2021","journal-title":"Weekly Epidemiological Record."},{"issue":"9","key":"pntd.0012167.ref017","doi-asserted-by":"crossref","first-page":"2009","DOI":"10.1007\/s00216-020-02490-2","article-title":"PCR inhibition in qPCR, dPCR and MPS\u2014mechanisms and solutions","volume":"412","author":"M Sidstedt","year":"2020","journal-title":"Anal Bioanal Chem"},{"key":"pntd.0012167.ref018","article-title":"Highly specific and sensitive detection of Yersinia pestis by portable Cas12a-UPTLFA platform.","volume":"12","author":"Y You","year":"2021","journal-title":"Front Microbiol."},{"key":"pntd.0012167.ref019","doi-asserted-by":"crossref","first-page":"114344","DOI":"10.1016\/j.bios.2022.114344","article-title":"Emerging digital PCR technology in precision medicine","volume":"211","author":"L Zhang","year":"2022","journal-title":"Biosens Bioelectron"},{"key":"pntd.0012167.ref020","doi-asserted-by":"crossref","first-page":"750","DOI":"10.1016\/j.ijbiomac.2021.06.132","article-title":"Digital PCR for accurate quantification of pathogens: principles, applications, challenges and future prospects","volume":"184","author":"S Lei","year":"2021","journal-title":"Int J Biol Macromol"},{"issue":"1","key":"pntd.0012167.ref021","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1186\/s13007-014-0042-6","article-title":"Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples.","volume":"10","author":"N Ra\u010dki","year":"2014","journal-title":"Plant Methods"},{"issue":"4","key":"pntd.0012167.ref022","doi-asserted-by":"crossref","first-page":"e0424922","DOI":"10.1128\/spectrum.04249-22","article-title":"Detection and quantification of Klebsiella pneumoniae in fecal samples using digital droplet pcr in comparison with real-time PCR.","volume":"11","author":"J Feng","year":"2023","journal-title":"Microbiol Spectr."},{"key":"pntd.0012167.ref023","article-title":"Development and evaluation of a multiplex droplet digital polymerase chain reaction method for simultaneous detection of five biothreat pathogens","volume":"13","author":"Y Du","year":"2022","journal-title":"Front Microbiol"},{"issue":"15","key":"pntd.0012167.ref024","doi-asserted-by":"crossref","first-page":"5147","DOI":"10.1128\/JB.186.15.5147-5152.2004","article-title":"Genetics of metabolic variations between Yersinia pestis biovars and the proposal of a new biovar, microtus","volume":"186","author":"D Zhou","year":"2004","journal-title":"J Bacteriol"},{"issue":"2","key":"pntd.0012167.ref025","doi-asserted-by":"crossref","first-page":"368","DOI":"10.4161\/hv.27060","article-title":"Yersinia pestis biovar Microtus strain 201, an avirulent strain to humans, provides protection against bubonic plague in rhesus macaques.","volume":"10","author":"Q Zhang","year":"2013","journal-title":"Hum Vaccin Immunother"},{"issue":"3","key":"pntd.0012167.ref026","doi-asserted-by":"crossref","first-page":"e629","DOI":"10.1371\/journal.pntd.0000629","article-title":"Ambient stable quantitative PCR reagents for the detection of Yersinia pestis.","volume":"4","author":"S Qu","year":"2010","journal-title":"PLoS Negl Trop Dis"},{"issue":"4","key":"pntd.0012167.ref027","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1111\/j.1472-765X.2008.02426.x","article-title":"Curing of four different plasmids in Yersinia pestis using plasmid incompatibility","volume":"47","author":"B Ni","year":"2008","journal-title":"Lett Appl Microbiol"},{"key":"pntd.0012167.ref028","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1016\/j.jviromet.2014.02.020","article-title":"Low copy target detection by droplet digital PCR through application of a novel open access bioinformatic pipeline, \u2018definetherain\u2019.","volume":"202","author":"M Jones","year":"2014","journal-title":"J Virol Methods"},{"issue":"4","key":"pntd.0012167.ref029","doi-asserted-by":"crossref","DOI":"10.1128\/spectrum.00704-22","article-title":"Absolute quantification of viable but nonculturable vibrio cholerae using droplet digital PCR with oil-enveloped bacterial cells.","volume":"10","author":"S Zhao","year":"2022","journal-title":"Microbiol Spectr."},{"issue":"6855","key":"pntd.0012167.ref030","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1038\/35097083","article-title":"Genome sequence of Yersinia pestis, the causative agent of plague","volume":"413","author":"J Parkhill","year":"2001","journal-title":"Nature"},{"issue":"1","key":"pntd.0012167.ref031","doi-asserted-by":"crossref","DOI":"10.1186\/s13104-015-1525-x","article-title":"The pla gene, encoding plasminogen activator, is not specific to Yersinia pestis.","volume":"8","author":"S H\u00e4nsch","year":"2015","journal-title":"BMC Res Notes."},{"issue":"6","key":"pntd.0012167.ref032","doi-asserted-by":"crossref","first-page":"834","DOI":"10.1111\/1749-4877.12546","article-title":"No evidence for enzootic plague within black-tailed prairie dog (Cynomys ludovicianus) populations.","volume":"16","author":"RE Colman","year":"2021","journal-title":"Integr Zool"},{"issue":"10","key":"pntd.0012167.ref033","doi-asserted-by":"crossref","first-page":"e1105","DOI":"10.1002\/mbo3.1105","article-title":"Pentaplex real-time PCR for differential detection of Yersinia pestis and Y. pseudotuberculosis and application for testing fleas collected during plague epizootics","volume":"9","author":"Y Bai","year":"2020","journal-title":"Microbiologyopen"},{"issue":"8","key":"pntd.0012167.ref034","doi-asserted-by":"crossref","first-page":"e0009558","DOI":"10.1371\/journal.pntd.0009558","article-title":"Genetic source tracking of human plague cases in Inner Mongolia-Beijing, 2019.","volume":"15","author":"J Li","year":"2021","journal-title":"PLoS Negl Trop Dis"}],"updated-by":[{"DOI":"10.1371\/journal.pntd.0012167","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2024,5,15]],"date-time":"2024-05-15T00:00:00Z","timestamp":1715731200000}}],"container-title":["PLOS Neglected Tropical Diseases"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pntd.0012167","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,5,15]],"date-time":"2024-05-15T17:52:10Z","timestamp":1715795530000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pntd.0012167"}},"subtitle":[],"editor":[{"given":"Vladimir L.","family":"Motin","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2024,5,3]]},"references-count":34,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2024,5,3]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pntd.0012167","relation":{"new_version":[{"id-type":"doi","id":"10.1371\/journal.pntd.0012167","asserted-by":"object"}]},"ISSN":["1935-2735"],"issn-type":[{"value":"1935-2735","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,5,3]]}}}