An improved RT-qPCR method for direct quantification of enveloped RNA viruses
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 b...
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| Format: | Article |
| Language: | English |
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Elsevier
2022-01-01
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| Series: | MethodsX |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2215016122001182 |
| _version_ | 1811201304263917568 |
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| author | Pavlina Gregorova Minna-Maria K. Heinonen L. Peter Sarin |
| author_facet | Pavlina Gregorova Minna-Maria K. Heinonen L. Peter Sarin |
| author_sort | Pavlina Gregorova |
| collection | DOAJ |
| description | 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. • One-step direct RT-qPCR quantification of phage phi6 virus without prior RNA isolation. • Reduced reaction volume for sustainable and cost-effective analysis. |
| first_indexed | 2024-04-12T02:18:42Z |
| format | Article |
| id | doaj.art-20ba3fbe590d43afa5416293f37c7677 |
| institution | Directory Open Access Journal |
| issn | 2215-0161 |
| language | English |
| last_indexed | 2024-04-12T02:18:42Z |
| publishDate | 2022-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | MethodsX |
| spelling | doaj.art-20ba3fbe590d43afa5416293f37c76772022-12-22T03:52:10ZengElsevierMethodsX2215-01612022-01-019101737An improved RT-qPCR method for direct quantification of enveloped RNA virusesPavlina Gregorova0Minna-Maria K. Heinonen1L. Peter Sarin2RNAcious Laboratory, Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, FinlandRNAcious Laboratory, Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, FinlandCorresponding author.; RNAcious Laboratory, Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, FinlandReverse 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. • One-step direct RT-qPCR quantification of phage phi6 virus without prior RNA isolation. • Reduced reaction volume for sustainable and cost-effective analysis.http://www.sciencedirect.com/science/article/pii/S2215016122001182RT-qPCR for quantification of RNA phages |
| spellingShingle | Pavlina Gregorova Minna-Maria K. Heinonen L. Peter Sarin An improved RT-qPCR method for direct quantification of enveloped RNA viruses MethodsX RT-qPCR for quantification of RNA phages |
| title | An improved RT-qPCR method for direct quantification of enveloped RNA viruses |
| title_full | An improved RT-qPCR method for direct quantification of enveloped RNA viruses |
| title_fullStr | An improved RT-qPCR method for direct quantification of enveloped RNA viruses |
| title_full_unstemmed | An improved RT-qPCR method for direct quantification of enveloped RNA viruses |
| title_short | An improved RT-qPCR method for direct quantification of enveloped RNA viruses |
| title_sort | improved rt qpcr method for direct quantification of enveloped rna viruses |
| topic | RT-qPCR for quantification of RNA phages |
| url | http://www.sciencedirect.com/science/article/pii/S2215016122001182 |
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