{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,29]],"date-time":"2026-10-29T16:40:50Z","timestamp":1793292050002,"version":"4.5.0"},"reference-count":51,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2025,5,23]],"date-time":"2025-05-23T00:00:00Z","timestamp":1747958400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100011102","name":"Seventh Framework Programme","doi-asserted-by":"publisher","award":["312631"],"award-info":[{"award-number":["312631"]}],"id":[{"id":"10.13039\/100011102","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100010790","name":"Erasmus+","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100010790","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p><jats:italic>Staphylococcus<\/jats:italic> (<jats:italic>S.<\/jats:italic>) <jats:italic>aureus<\/jats:italic> is a prominent foodborne pathogen that can cause food poisoning due to its staphylococcal toxins. Controlling the viable levels of <jats:italic>S. aureus<\/jats:italic> is crucial for ensuring food safety. The detection of <jats:italic>S. aureus<\/jats:italic> during routine quality control is still primarily conducted using traditional culture-based methods, which are time-consuming and unable to detect viable but non-culturable cells. Viability PCR (vPCR) \u2013 a combination of traditional (or quantitative) PCR with photo-reactive DNA-intercalating dye(s) \u2013 has been introduced as an alternative to detect viable cells by excluding those with compromised membranes using molecular methods. Despite the success of the vPCR methodology, avoiding false-positive results remains a significant challenge. To enhance the accuracy of vPCR results for <jats:italic>S. aureus<\/jats:italic>, several approaches have been proposed by various researchers in the past decade; however, complete PCR signal suppression of dead cells has not been achieved. In this study, we developed a strategy to detect only viable <jats:italic>S. aureus<\/jats:italic> cells by combining double PMA treatment with a low PMA concentration and performing a tube change between the last dark incubation and light exposure to improve the vPCR protocol. For pure cultures, the optimized protocol was able to completely suppress DNA signals from 5.0\u2009\u00d7\u200910<jats:sup>7<\/jats:sup> dead cells in a final reaction volume of 200 \u00b5l. For artificially contaminated food samples with such a high dead cell count, complete PCR signal reduction was observed in ground pepper, - oregano, and infant milk powder, while ground paprika, - allspice, and - pork exhibited PCR signals close to the detection limit. To simulate conditions in real samples, we artificially contaminated ground paprika, - pork, and milk powder with a low number of viable cells (~1.9 cfu\/ml) and a high number of heat-inactivated <jats:italic>S. aureus<\/jats:italic> (~4.8\u2009\u00d7\u200910\u2076 cells\/ml). The results showed that the optimized protocol is effective in detecting only the desired live cells, even in the presence of a high dead cell count. Our findings highlight that vPCR can be an accurate and reliable method with strong potential for high-throughput detection of live <jats:italic>S. aureus<\/jats:italic> cells in certain food matrices.<\/jats:p>","DOI":"10.1371\/journal.pone.0324819","type":"journal-article","created":{"date-parts":[[2025,5,23]],"date-time":"2025-05-23T17:40:10Z","timestamp":1748022010000},"page":"e0324819","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":8,"title":["Optimization of the viability PCR for accurate detection of Staphylococcus aureus in food samples"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3025-973X","authenticated-orcid":true,"given":"Mai","family":"Dinh Thanh","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gemma","family":"Agust\u00ed","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anneluise","family":"Mader","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesc","family":"Codony","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,5,23]]},"reference":[{"issue":"12","key":"pone.0324819.ref001","doi-asserted-by":"crossref","first-page":"751","DOI":"10.1016\/S1473-3099(05)70295-4","article-title":"The role of nasal carriage in Staphylococcus aureus infections","volume":"5","author":"HFL Wertheim","year":"2005","journal-title":"Lancet Infect Dis"},{"issue":"3","key":"pone.0324819.ref002","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1128\/CMR.00134-14","article-title":"Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management","volume":"28","author":"SYC Tong","year":"2015","journal-title":"Clin Microbiol Rev"},{"issue":"4","key":"pone.0324819.ref003","doi-asserted-by":"crossref","first-page":"815","DOI":"10.1111\/j.1574-6976.2011.00311.x","article-title":"Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation","volume":"36","author":"J-A Hennekinne","year":"2012","journal-title":"FEMS Microbiol Rev"},{"issue":"12","key":"pone.0324819.ref004","article-title":"The European union one health 2023 zoonoses report","volume":"22","author":"European Food Safety Authority (EFSA), European Centre for Disease Prevention and Control (ECDC)","year":"2024","journal-title":"EFSA J"},{"issue":"5","key":"pone.0324819.ref005","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1016\/S0963-9969(02)00194-1","article-title":"Microbiological quality of some retail spices in India","volume":"36","author":"M Banerjee","year":"2003","journal-title":"Food Res Int"},{"issue":"4","key":"pone.0324819.ref006","doi-asserted-by":"crossref","first-page":"364","DOI":"10.1007\/s11130-010-0186-0","article-title":"Assessment of the microbiological safety of dried spices and herbs commercialized in Spain","volume":"65","author":"I Sospedra","year":"2010","journal-title":"Plant Foods Hum Nutr"},{"issue":"3","key":"pone.0324819.ref007","doi-asserted-by":"crossref","first-page":"624","DOI":"10.1099\/00221287-19-3-624","article-title":"The effect of residual water on the survival of dried bacteria during storage","volume":"19","author":"WJ Scott","year":"1958","journal-title":"J Gen Microbiol"},{"key":"pone.0324819.ref008","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.foodcont.2016.12.027","article-title":"Tenacity of Bacillus cereus and Staphylococcus aureus in dried spices and herbs","volume":"83","author":"M Dinh Thanh","year":"2018","journal-title":"Food Control"},{"key":"pone.0324819.ref009","unstructured":"International Organization for Standardization. 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