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AuthorHasan, Mohammad Rubayet
AuthorMirza, Faheem
AuthorAl-Hail, Hamad
AuthorSundararaju, Sathyavathi
AuthorXaba, Thabisile
AuthorIqbal, Muhammad
AuthorAlhussain, Hashim
AuthorYassine, Hadi Mohamad
AuthorPerez-Lopez, Andres
AuthorTang, Patrick
Available date2020-08-30T05:20:31Z
Publication Date2020-07-24
Publication NamePLOS ONE
Identifierhttp://dx.doi.org/10.1371/journal.pone.0236564
CitationHasan MR, Mirza F, Al-Hail H, Sundararaju S, Xaba T, Iqbal M, et al. (2020) Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA. PLoS ONE 15(7): e0236564. https://doi.org/10.1371/journal.pone.0236564
URIhttp://hdl.handle.net/10576/15854
AbstractTo circumvent the limited availability of RNA extraction reagents, we aimed to develop a protocol for direct RT-qPCR to detect SARS-CoV-2 in nasopharyngeal swabs without RNA extraction. Nasopharyngeal specimens positive for SARS-CoV-2 and other coronaviruses collected in universal viral transport (UVT) medium were pre-processed by several commercial and laboratory-developed methods and tested by RT-qPCR assays without RNA extraction using different RT-qPCR master mixes. The results were compared to that of standard approach that involves RNA extraction. Incubation of specimens at 65°C for 10 minutes along with the use of TaqPath™ 1-Step RT-qPCR Master Mix provides higher analytical sensitivity for detection of SARS-CoV-2 RNA than many other conditions tested. The optimized direct RT-qPCR approach demonstrated a limit of detection of 6.6x103 copy/ml and high reproducibility (co-efficient of variation = 1.2%). In 132 nasopharyngeal specimens submitted for SARS-CoV-2 testing, the sensitivity, specificity and accuracy of our optimized approach were 95%, 99% and 98.5%, respectively, with reference to the standard approach. Also, the RT-qPCR CT values obtained by the two methods were positively correlated (Pearson correlation coefficient r = 0.6971, p = 0.0013). The rate of PCR inhibition by the direct approach was 8% compared to 9% by the standard approach. Our simple approach to detect SARS-CoV-2 RNA by direct RT-qPCR may help laboratories continue testing for the virus despite reagent shortages or expand their testing capacity in resource limited settings.
Languageen
PublisherPublic Library of Science
SubjectSARS-CoV-2
Betacoronavirus
Clinical Laboratory Techniques/methods
Coronavirus Infections/diagnosis
Humans
Nasopharynx/virology
Pandemics
TitleDetection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA.
TypeArticle
Issue Number7
Volume Number15
ESSN1932-6203
dc.accessType Open Access


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