{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,18]],"date-time":"2026-05-18T11:18:46Z","timestamp":1779103126866,"version":"3.51.4"},"reference-count":25,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2021,5,18]],"date-time":"2021-05-18T00:00:00Z","timestamp":1621296000000},"content-version":"vor","delay-in-days":17,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2021,5]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Since December 2019, SARS\u2010CoV\u20102 has spread extensively throughout the world, with more than 117 million reported cases and 2.6 million deaths (Johns Hopkins coronavirus resource center, <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"https:\/\/coronavirus.jhu.edu\/map.html\">https:\/\/coronavirus.jhu.edu\/map.html<\/jats:ext-link>). Detecting the virus is the first step in diagnosing the infection, followed by quarantine to prevent transmission. Nasopharyngeal\/oropharyngeal swabs (NP\/OP) and saliva are two specimen types that are most often analyzed to detect SARS\u2010CoV\u20102 by molecular tests that detect viral RNA or by antigen\/antibody tests that detect viral proteins and\/or the host immune response against the virus. Compared to antigen\/antibody tests, molecular tests are highly sensitive and specific for detecting the virus. A significant drawback is that specimen collection requirements are specific to each test and cannot be interchanged with another test. Some tests are qualified to be used on NP swabs or saliva, but not both specimen types. Even with NP swabs, a test may be qualified to detect the virus only with swabs collected in viral transport medium (VTM) but not in other media. These restrictive pre\u2010analytic steps are disadvantageous in that a lab would have to develop and validate different tests for SARS\u2010CoV\u20102 depending on the specimen type and collection media, with added setup cost, infrastructure, and training requirements. To overcome these problems, we developed and validated a cost\u2010effective multiplex reverse\u2010transcription real\u2010time PCR assay that can be used to detect SARS\u2010CoV\u20102 in different specimen types. The assay is highly sensitive and specific, can be used to detect the virus in saliva as well as NP swabs collected in different media such as VTM, saline, and commercial preservative fluid, and serves as one test for all applications. The protocol also describes an optimal laboratory setup and unidirectional workflow for detecting SARS\u2010CoV\u20102 by RT\u2010qPCR. \u00a9 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Manual viral nucleic acid extraction from NP\/OP swabs collected in different media, and from saliva<\/jats:p><jats:p><jats:bold>Alternate Protocol 1<\/jats:bold>: Low\u2010throughput automated extraction on the Qiagen EZ1 Advanced XL machine (1\u201014 samples)<\/jats:p><jats:p><jats:bold>Alternate Protocol 2<\/jats:bold>: High\u2010throughput automated extraction on the Kingfisher Flex machine (1\u201096 samples)<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Multiplex RT\u2010qPCR protocol to detect SARS\u2010CoV\u20102<\/jats:p><jats:p><jats:bold>Alternate Protocol 3<\/jats:bold>: Multiplex one\u2010step RT\u2010qPCR protocol to detect SARS\u2010CoV\u20102 with S and E gene probes labeled with the same fluorochrome<\/jats:p>","DOI":"10.1002\/cpz1.145","type":"journal-article","created":{"date-parts":[[2021,5,18]],"date-time":"2021-05-18T19:09:32Z","timestamp":1621364972000},"source":"Crossref","is-referenced-by-count":5,"title":["A Multiplex One\u2010Step RT\u2010qPCR Protocol to Detect SARS\u2010CoV\u20102 in NP\/OP Swabs and Saliva"],"prefix":"10.1002","volume":"1","author":[{"given":"Joshua","family":"Bland","sequence":"first","affiliation":[{"name":"Department of Pathology University of Illinois at Chicago  Chicago Illinois"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ashley","family":"Kavanaugh","sequence":"additional","affiliation":[{"name":"Department of Pathology University of Illinois at Chicago  Chicago Illinois"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lenny K.","family":"Hong","sequence":"additional","affiliation":[{"name":"Department of Pathology University of Illinois at Chicago  Chicago Illinois"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Omar","family":"Perez","sequence":"additional","affiliation":[{"name":"Department of Pathology University of Illinois at Chicago  Chicago Illinois"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shrihari S.","family":"Kadkol","sequence":"additional","affiliation":[{"name":"Department of Pathology University of Illinois at Chicago  Chicago 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