{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:26:26Z","timestamp":1760059586214,"version":"build-2065373602"},"reference-count":35,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2025,6,21]],"date-time":"2025-06-21T00:00:00Z","timestamp":1750464000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Iowa Livestock Health Advisory Council (ILHAC)-2024","award":["PG114003"],"award-info":[{"award-number":["PG114003"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Veterinary Sciences"],"abstract":"<jats:p>Tonsil oral scrubbing (TOSc) is a simple and practical method recently developed for detecting PRRSV RNA in sows. This study evaluated how pooling and freeze-thaw procedures affect PRRSV RT-rtPCR results in TOSc samples. To assess pooling effects, 22 PRRSV-positive samples were divided into three Ct categories and diluted with negative TOSc samples at various ratios before testing. To assess freeze-thaw effects, 90 TOSc samples were split into two equal aliquots: one tested immediately, and the other tested after one freeze-thaw cycle. Results showed that PRRSV RNA detection probability decreased and Ct values increased as dilution (pooling) ratios rose. Samples with Ct values &gt; 35 had significant drops in probability of detection beginning at a 1:3 dilution (\u226471%). In contrast, samples with Ct values &lt;35 maintained a high probability of detection until 1:10 dilution (\u226585%). The freeze-thawed samples showed significantly higher Ct values and numerically lower probability of detection compared to non-freeze-thawed ones. In conclusion, pooling and freeze-thaw negatively impact PRRSV RNA detection in TOSc samples. Pooling may be acceptable for samples with a Ct value &lt; 35 for up to 1:10 dilution, but should be avoided when the Ct value &gt; 35. Similarly, the freeze-thaw process should be minimized during TOSc sample handling.<\/jats:p>","DOI":"10.3390\/vetsci12070607","type":"journal-article","created":{"date-parts":[[2025,6,23]],"date-time":"2025-06-23T09:08:44Z","timestamp":1750669724000},"page":"607","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Effect of Pooling and Freeze-Thaw Processes on the PRRSV RNA Detection in TOSc Samples"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-5625-0420","authenticated-orcid":false,"given":"Peng","family":"Li","sequence":"first","affiliation":[{"name":"College of Veterinary Medicine, Iowa State University, Ames, IA 50010, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Onyekachukwu H.","family":"Osemeke","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Iowa State University, Ames, IA 50010, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-3440-621X","authenticated-orcid":false,"given":"Thinh","family":"Tran Pham Tien","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Iowa State University, Ames, IA 50010, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-0686-1959","authenticated-orcid":false,"given":"Ching-Yang","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Iowa State University, Ames, IA 50010, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Angie","family":"Supple","sequence":"additional","affiliation":[{"name":"Eichelberger Farms, Wayland, IA 52654, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3638-7721","authenticated-orcid":false,"given":"Marcelo N.","family":"Almeida","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Iowa State University, Ames, IA 50010, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7788-9942","authenticated-orcid":false,"given":"Daniel C. L.","family":"Linhares","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Iowa State University, Ames, IA 50010, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1186\/s13567-016-0391-4","article-title":"Review on the transmission porcine reproductive and respiratory syndrome virus between pigs and farms and impact on vaccination","volume":"47","author":"Pileri","year":"2016","journal-title":"Vet. 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