An improved RT-qPCR method for direct quantification of enveloped RNA viruses

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Gregorova , P , Heinonen , M-M K & Sarin , P 2022 , ' An improved RT-qPCR method for direct quantification of enveloped RNA viruses ' , MethodsX , vol. 9 , 101737 . https://doi.org/10.1016/j.mex.2022.101737

Title: An improved RT-qPCR method for direct quantification of enveloped RNA viruses
Author: Gregorova, Pavlina; Heinonen, Minna-Maria Kristiina; Sarin, Peter
Contributor organization: RNAcious laboratory
Molecular and Integrative Biosciences Research Programme
Date: 2022-06-01
Language: eng
Number of pages: 9
Belongs to series: MethodsX
ISSN: 2215-0161
DOI: https://doi.org/10.1016/j.mex.2022.101737
URI: http://hdl.handle.net/10138/344424
Abstract: Reverse transcription quantitative PCR (RT-qPCR) has emerged as the gold standard for virus detection and quantification, being utilized in numerous diagnostic and research applications. However, the direct detection of viruses has so far posed a challenge as the viral genome is often encapsidated by a proteinaceous layer surrounded by a lipid envelope. This necessitates an additional and undesired RNA extraction step prior to RT-qPCR amplification. To circumvent this limitation, we have developed a direct RT-qPCR method for the detection of RNA viruses. In our method, we provide a proof-of-concept using phage phi6, a safe-to-use proxy for pathogenic enveloped RNA viruses that is commonly utilized in e.g. aerosolization studies. First, the phage phi6 envelope is removed by 1% chloroform treatment and the virus is then directly quantified by RT-qPCR. To identify false negative results, firefly luciferase is included as a synthetic external control. Thanks to the duplex format, our direct RT-qPCR method reduces the reagents needed and provides an easy to implement and broadly applicable, fast, and cost-effective tool for the quantitative analysis of enveloped RNA viruses.center dot One-step direct RT-qPCR quantification of phage phi6 virus without prior RNA isolation.center dot Reduced reaction volume for sustainable and cost-effective analysis.(c) 2022 The Author(s). Published by Elsevier B.V.This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )
Subject: 1182 Biochemistry, cell and molecular biology
RT-qPCR
direct detection
RNA isolation-free
11832 Microbiology and virology
RNA virus
phage phi 6
RT-qPCR
Direct quantification
Bacteriophage
Phi6
RNA virus
RNA-dependent RNA polymerase
SARS-COV-2
Peer reviewed: Yes
Rights: cc_by
Usage restriction: openAccess
Self-archived version: publishedVersion
Funder: Business Finland
Grant number:


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