{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,28]],"date-time":"2026-07-28T05:20:40Z","timestamp":1785216040197,"version":"3.55.0"},"reference-count":60,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,2,18]],"date-time":"2026-02-18T00:00:00Z","timestamp":1771372800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Salmonella remains a leading cause of foodborne illness worldwide, with poultry products representing a major source of human exposure, underscoring the need for rapid and reliable detection methods. Although qPCR offers sensitive and timely pathogen detection, assay performance is highly dependent on sample preparation efficiency and nucleic acid purity, particularly in complex food matrices. In this study, we systematically optimized the sample preparation workflow of a SYBR Green based qPCR assay for enrichment-free detection of Salmonella enterica in poultry. Multiple lysis chemistries, incubation times, DNA extraction methods, centrifugation strategies, inoculum sources, and magnetic nanoparticle (MNP) assisted workflows were evaluated using phosphate-buffered saline and chicken rinsate matrices. Among the conditions tested, lysis with 20 \u00b5L Proteinase K and 400 \u00b5L PrepMan\u2122 for 20 min produced the lowest and most consistent Cq values. Although Promega Wizard\u00ae produced slightly lower mean Cq values than PrepMan\u2122, statistical analysis showed no significant differences between extraction methods or centrifugation protocols, indicating comparable overall performance. Broth-derived inocula yielded earlier and more reproducible Cq values than colony-derived preparations. In contrast, inclusion of MNP processing resulted in higher Cq values in both matrices compared to the non-MNP workflow. Overall, these findings demonstrate that optimized lysis, extraction, and centrifugation workflows enhances the consistency and analytical reliability of direct qPCR detection of Salmonella in poultry matrices, supporting laboratory-based rapid detection applications.<\/jats:p>","DOI":"10.3390\/pathogens15020229","type":"journal-article","created":{"date-parts":[[2026,2,18]],"date-time":"2026-02-18T18:16:31Z","timestamp":1771438591000},"page":"229","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Optimizing Lysis and Extraction Workflows for Enrichment-Free qPCR Detection of Salmonella\u00a0enterica in Poultry Matrices"],"prefix":"10.3390","volume":"15","author":[{"given":"Rejoice","family":"Nyarku","sequence":"first","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emmanuel","family":"Kuufire","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kingsley E.","family":"Bentum","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Viona","family":"Osei","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Asmaa","family":"Elrefaey","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tyric","family":"James","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yilkal","family":"Woube","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0027-176X","authenticated-orcid":false,"given":"Evangelyn","family":"Alocilja","sequence":"additional","affiliation":[{"name":"Global Alliance for Rapid Diagnostics, Michigan State University, East Lansing, MI 48824, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9722-5318","authenticated-orcid":false,"given":"Temesgen","family":"Samuel","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Woubit","family":"Abebe","sequence":"additional","affiliation":[{"name":"Center for Food Animal Health, Food Safety and Defense, Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36088, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"8899596","DOI":"10.1155\/2023\/8899596","article-title":"Prevention and control of human Salmonella enterica infections: Implications for food safety","volume":"2023","author":"Mkangara","year":"2023","journal-title":"Int. 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