{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,30]],"date-time":"2026-08-30T00:40:46Z","timestamp":1788050446749,"version":"build-2784847793"},"reference-count":33,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,7,28]],"date-time":"2022-07-28T00:00:00Z","timestamp":1658966400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>Biothreat agents pose a huge threat to human and public health, necessitating the development of rapid and highly sensitive detection approaches. This study establishes a multiplex droplet digital polymerase chain reaction (ddPCR) method for simultaneously detecting five high-risk bacterial biothreats: <jats:italic>Yersinia pestis<\/jats:italic>, <jats:italic>Bacillus anthracis<\/jats:italic>, <jats:italic>Brucella<\/jats:italic> spp., <jats:italic>Burkholderia pseudomallei<\/jats:italic>, and <jats:italic>Francisella tularensis<\/jats:italic>. Unlike conventional multiplex real-time PCR (qPCR) methods, the multiplex ddPCR assay was developed using two types of probe fluorophores, allowing the assay to perform with a common two-color ddPCR system. After optimization, the assay performance was evaluated, showing a lower limit of detection (LOD) (0.1\u20131.0 pg\/\u03bcL) and good selectivity for the five bacteria targets. The multiplex assay\u2019s ability to simultaneously detect two or more kinds of targets in a sample was also demonstrated. The assay showed strong sample tolerance when testing simulated soil samples; the LOD for bacteria in soil was 2 \u00d7 10<jats:sup>2<\/jats:sup>\u20132 \u00d7 10<jats:sup>3<\/jats:sup> colony-forming unit (CFU)\/100 mg soil (around 5\u201350 CFU\/reaction), which was 10-fold lower than that of the single-target qPCR method. When testing simulated soil samples at bacterial concentrations of 2 \u00d7 10<jats:sup>3<\/jats:sup>\u20132 \u00d7 10<jats:sup>4<\/jats:sup> CFU\/100 mg soil, the assay presented a higher sensitivity (100%, 35\/35) than that of the qPCR method (65.71%, 23\/35) and a good specificity (100%, 15\/15). These results suggest that the developed 5-plex ddPCR method is more sensitive than conventional qPCR methods and is potentially suitable for rapidly detecting or screening the five selected bacterial biothreats in suspicious samples.<\/jats:p>","DOI":"10.3389\/fmicb.2022.970973","type":"journal-article","created":{"date-parts":[[2022,7,28]],"date-time":"2022-07-28T06:58:03Z","timestamp":1658991483000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":28,"title":["Development and evaluation of a multiplex droplet digital polymerase chain reaction method for simultaneous detection of five biothreat pathogens"],"prefix":"10.3389","volume":"13","author":[{"given":"Yipu","family":"Du","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":"Junyan","family":"Jin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liting","family":"Xiao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhulin","family":"Sun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yafang","family":"Tan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pingping","family":"Zhang","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":"Yong","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yajun","family":"Song","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2022,7,28]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","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":"Banada","year":"2019","journal-title":"J. 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