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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