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Screening for the SARS-CoV-2 virus has been a key part of disease mitigation while the world continues to move forward, and lessons learned will benefit disease detection beyond COVID-19. Saliva specimen collection offers a less invasive, time- and cost-effective alternative to standard nasopharyngeal swabs. We optimized two different methods of saliva sample processing for RT-qPCR testing. Two methods were optimized to provide two cost-efficient ways to do testing for a minimum of four samples by pooling in a 2.0\u00a0mL tube and decrease the need for more highly trained personnel. Acid-pH-based RNA extraction method can be done without the need for expensive kits. Direct Lysis is a quick one-step reaction that can be applied quickly. Our optimized Acid-pH and Direct Lysis protocols are reliable and reproducible, detecting the beta-2 microglobulin (<jats:italic>B2M<\/jats:italic>) mRNA in saliva as an internal control from 97 to 96.7% of samples, respectively. The cycle threshold (Ct) values for<jats:italic>B2M<\/jats:italic>were significantly higher in the Direct Lysis protocol than in the Acid-pH protocol. The limit of detection for<jats:italic>N1<\/jats:italic>gene was higher in Direct Lysis at\u2009\u2264\u20095 copies\/\u03bcL than Acid-pH. Saliva samples collected over the course of several days from two COVID-positive individuals demonstrated Ct values for N1 that were consistently higher from Direct Lysis compared to Acid-pH. Collectively, this work supports that each of these techniques can be used to screen for SARS-CoV-2 in saliva for a cost-effective screening platform.<\/jats:p>","DOI":"10.1038\/s41598-024-54183-w","type":"journal-article","created":{"date-parts":[[2024,3,25]],"date-time":"2024-03-25T08:01:57Z","timestamp":1711353717000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Evaluating and optimizing Acid-pH and Direct Lysis RNA extraction for SARS-CoV-2 RNA detection in whole saliva"],"prefix":"10.1038","volume":"14","author":[{"given":"Brayden","family":"LaBute","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jackie","family":"Fong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Farinaz","family":"Ziaee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert","family":"Gombar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mathew","family":"Stover","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Terry","family":"Beaudin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Badalova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiudi","family":"Geng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryland","family":"Corchis-Scott","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana","family":"Podadera","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kyle","family":"Lago","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"ZhenHuan","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fievel","family":"Lim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Felix","family":"Chiu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Minghua","family":"Fu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaofeng","family":"Nie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuanmin","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Corrina","family":"Quan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caroline","family":"Hamm","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"R. 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