{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T13:04:24Z","timestamp":1790427864983,"version":"4.1.0"},"reference-count":34,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,11,2]],"date-time":"2021-11-02T00:00:00Z","timestamp":1635811200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:p>Many bacterial species, including <jats:italic>Vibrio cholerae<\/jats:italic> (the pathogen that causes cholera), enter a physiologically viable but non-culturable (VBNC) state at low temperature or in conditions of low nutrition; this is a survival strategy to resist environmental stress. Identification, detection, and differentiation of VBNC cells and nonviable cells are essential for both microbiological study and disease surveillance\/control. Enumeration of VBNC cells requires an accurate method. Traditional counting methods do not allow quantification of VBNC cells because they are not culturable. Morphology-based counting cannot distinguish between live and dead cells. A bacterial cell possesses one copy of the chromosome. Hence, counting single-copy genes on the chromosome is a suitable approach to count bacterial cells. In this study, we developed quantitative PCR-based methods, including real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR), to enumerate VBNC <jats:italic>V. cholerae<\/jats:italic> cells by counting the numbers of single-copy genes in samples during VBNC-state development. Propidium monoazide (PMA) treatment was incorporated to distinguish dead cells from viable cells. Both PCR methods could be used to quantify the number of DNA copies\/mL and determine the proportion of dead cells (when PMA was used). The methods produced comparable counts using three single-copy genes (VC1376, <jats:italic>thyA<\/jats:italic>, and <jats:italic>recA<\/jats:italic>). However, ddPCR showed greater accuracy and sensitivity than qPCR. ddPCR also allows direct counting without the need to establish a standard curve. Our study develops a PMA-ddPCR method as a new tool to quantify VBNC cells of <jats:italic>V. cholerae<\/jats:italic>. The method can be extended to other bacterial species.<\/jats:p>","DOI":"10.3389\/fcimb.2021.753078","type":"journal-article","created":{"date-parts":[[2021,11,2]],"date-time":"2021-11-02T05:36:26Z","timestamp":1635831386000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":17,"title":["Enumeration of Viable Non-Culturable Vibrio cholerae Using Droplet Digital PCR Combined With Propidium Monoazide Treatment"],"prefix":"10.3389","volume":"11","author":[{"given":"Shuo","family":"Zhao","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingyun","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhe","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu","family":"Han","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Biao","family":"Kan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2021,11,2]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1371\/journal.pgen.1005145","article-title":"Proteolysis of Virulence Regulator ToxR Is Associated With Entry of Vibrio cholerae Into a Dormant State","volume":"7","author":"Almagro-Moreno","year":"2015","journal-title":"PLoS Genet."},{"key":"B2","doi-asserted-by":"publisher","first-page":"869","DOI":"10.1111\/j.1462-2920.2006.01206.x","article-title":"Gene Expression Profile of Vibrio cholerae in the Cold Stress Induced Viable But Non-Culturable State","volume":"9","author":"Asakura","year":"2007","journal-title":"Environ. 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