{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,21]],"date-time":"2026-04-21T08:20:10Z","timestamp":1776759610716,"version":"3.51.2"},"reference-count":48,"publisher":"Public Library of Science (PLoS)","issue":"2","license":[{"start":{"date-parts":[[2021,2,3]],"date-time":"2021-02-03T00:00:00Z","timestamp":1612310400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000011","name":"Howard Hughes Medical Institute","doi-asserted-by":"crossref","award":["CC34430"],"award-info":[{"award-number":["CC34430"]}],"id":[{"id":"10.13039\/100000011","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100001033","name":"Jane Coffin Childs Memorial Fund for Medical Research","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100001033","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. This study evaluates several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, isopropanol precipitation is shown to provide an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, direct addition of NP swab samples to RT-qPCRs is evaluated without an RNA extraction step. A simple, inexpensive swab collection solution suitable for direct addition is validated using contrived swab samples. Third, an open-source master mix for RT-qPCR is described that permits detection of viral RNA in NP swab samples with a limit of detection of approximately 50 RNA copies per reaction. Quantification cycle (Cq) values for purified RNA from 30 known positive clinical samples showed a strong correlation (r\n                    <jats:sup>2<\/jats:sup>\n                    = 0.98) between this homemade master mix and commercial TaqPath master mix. Lastly, end-point fluorescence imaging is found to provide an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, open-source methods has the potential to reduce the time and expense of COVID-19 testing.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0246647","type":"journal-article","created":{"date-parts":[[2021,2,3]],"date-time":"2021-02-03T14:29:42Z","timestamp":1612362582000},"page":"e0246647","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":35,"title":["Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection"],"prefix":"10.1371","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5189-4313","authenticated-orcid":true,"given":"Thomas G. W.","family":"Graham","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8602-3534","authenticated-orcid":true,"given":"Claire","family":"Dugast-Darzacq","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gina M.","family":"Dailey","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7532-8356","authenticated-orcid":true,"given":"Xammy Huu","family":"Wrynla","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4773-379X","authenticated-orcid":true,"given":"Erik","family":"Van Dis","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2157-3831","authenticated-orcid":true,"given":"Meagan N.","family":"Esbin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Abrar","family":"Abidi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sarah A.","family":"Stanley","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xavier","family":"Darzacq","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0539-8217","authenticated-orcid":true,"given":"Robert","family":"Tjian","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2021,2,3]]},"reference":[{"key":"pone.0246647.ref001","article-title":"\u2018It\u2019s Like Having No Testing\u2019: Coronavirus Test Results Are Still Delayed","volume":"7","author":"S Mervosh","year":"2020","journal-title":"New York Times"},{"key":"pone.0246647.ref002","article-title":"Test sensitivity is secondary to frequency and turnaround time for COVID-19 surveillance","author":"DB Larremore","year":"2020","journal-title":"medRxiv Prepr Serv Heal Sci"},{"key":"pone.0246647.ref003","article-title":"CDC 2019-novel coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel for emergency use only instructions for use","author":"Centers for Disease Control and Prevention","year":"2020","journal-title":"Atlanta"},{"key":"pone.0246647.ref004","article-title":"RNA Extraction Kits for COVID-19 Tests Are in Short Supply in US","author":"J. Akst","year":"2020","journal-title":"Sci"},{"key":"pone.0246647.ref005","doi-asserted-by":"crossref","first-page":"107","DOI":"10.5607\/en20009","article-title":"Development of a Laboratory-safe and Low-cost Detection Protocol for SARS-CoV-2 of the Coronavirus Disease 2019 (COVID-19)","volume":"29","author":"J Won","year":"2020","journal-title":"Exp Neurobiol"},{"key":"pone.0246647.ref006","article-title":"Fast SARS-CoV-2 detection protocol based on RNA precipitation and RT-qPCR in nasopharyngeal swab samples","author":"Xabier Guruceaga","year":"2020","journal-title":"medRxiv"},{"key":"pone.0246647.ref007","article-title":"A Fast and Accessible Method for the Isolation of RNA, DNA, and Protein to Facilitate the Detection of SARS-CoV-2","author":"Jose Carlos Ponce-Rojas","year":"2020","journal-title":"bioRxiv"},{"key":"pone.0246647.ref008","doi-asserted-by":"crossref","DOI":"10.1038\/s41598-020-73616-w","article-title":"A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR","author":"A Wozniak","year":"2020"},{"key":"pone.0246647.ref009","doi-asserted-by":"crossref","first-page":"771","DOI":"10.1261\/rna.076232.120","article-title":"Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection","volume":"26","author":"MN Esbin","year":"2020","journal-title":"RNA"},{"key":"pone.0246647.ref010","article-title":"Extraction-free COVID-19 (SARS-CoV-2) diagnosis by RT-PCR to increase capacity for national testing programmes during a pandemic","author":"PR Grant","year":"2020","journal-title":"bioRxiv"},{"key":"pone.0246647.ref011","article-title":"Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR","author":"I Smyrlaki","year":"2020","journal-title":"medRxiv"},{"key":"pone.0246647.ref012","article-title":"The potential use of unprocessed sample for RT-qPCR detection of COVID-19 without an RNA extraction step","author":"A Arumugam","year":"2020","journal-title":"bioRxiv"},{"key":"pone.0246647.ref013","article-title":"Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples","author":"J Alcoba-Florez","year":"2020","journal-title":"medRxiv"},{"key":"pone.0246647.ref014","article-title":"An alternative workflow for molecular detection of SARS-CoV-2\u2014escape from the NA extraction kit-shortage","author":"AS Fomsgaard","year":"2020","journal-title":"medRxiv"},{"key":"pone.0246647.ref015","article-title":"Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA","volume":"15","author":"MR Hasan","year":"2020","journal-title":"PLoS One"},{"key":"pone.0246647.ref016","article-title":"Multiplexed and extraction-free amplification for simplified SARS-CoV-2 RT-PCR tests","author":"SA Byrnes","year":"2020","journal-title":"medRxiv"},{"key":"pone.0246647.ref017","article-title":"DIRECT RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWABS WITHOUT AN RNA EXTRACTION STEP","author":"EA Bruce","year":"2020","journal-title":"bioRxiv"},{"key":"pone.0246647.ref018","first-page":"104423","article-title":"SARS-CoV-2 detection by direct rRT-PCR without RNA extraction","volume":"128","author":"N Merindol","journal-title":"J Clin Virol. 2020"},{"key":"pone.0246647.ref019","article-title":"A streamlined approach to rapidly detect SARS-CoV-2 infection, avoiding","author":"S Marzinotto","year":"2020","journal-title":"RNA extraction. medRxiv"},{"key":"pone.0246647.ref020","article-title":"SARS-CoV-2 detection from nasopharyngeal swab samples without","author":"C Beltr\u00e1n-Pavez","year":"2020","journal-title":"RNA extraction. bioRxiv"},{"key":"pone.0246647.ref021","article-title":"One-step RNA extraction for RT-qPCR detection of 2019-nCoV","author":"M Sentmanat","year":"2020","journal-title":"bioRxiv"},{"key":"pone.0246647.ref022","article-title":"Point-of-care testing for COVID-19 using SHERLOCK 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[cited 9 May 2020]. Available: https:\/\/openbioeconomy.org\/projects\/open-enzyme-collections\/"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0246647","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,8,27]],"date-time":"2025-08-27T13:49:53Z","timestamp":1756302593000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0246647"}},"subtitle":[],"editor":[{"given":"A. M.","family":"Abd El-Aty","sequence":"first","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2021,2,3]]},"references-count":48,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2021,2,3]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0246647","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2020.09.16.20193466","asserted-by":"object"}]},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,2,3]]}}}