Abstract
The pandemic coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread and high mortality rate. Detection and quantification of the (+) ssRNA virus, which has a genome size of 29,903 nucleotides, is commonly performed via reverse transcription quantitative polymerase chain reaction (RT-qPCR) targeting conserved sequences. Here, we describe a one-step RT-qPCR protocol for the quantitative detection of SARS-CoV-2 genomic RNA targeting M and RdRP genes, respectively, as well as active virus replication detecting subgenomic RNAs (sgRNA 4 and 8) that are formed by discontinuous transcription of the viral genome. Concomitantly, an input control targeting the human RNaseP gene (RPP30) was used in multiplex PCR to monitor the input of human nucleic acids. In vitro–transcribed RNA harboring the amplicon regions for M and RdRP regions served to set up a standard curve for absolute quantification.
In conclusion, the method described here allows for the detection and quantification of SARS-CoV-2 RNA isoforms for research by both using a probe-based or SYBR Green–based approach, but is also suitable for diagnostic purposes.
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Acknowledgments
The authors thank Denisa Bojkova, Jindrich Cinatl Jr., and Tuna Toptan-Grabmair for providing reagents and fruitful discussions. We are thankful for the numerous donations to the Goethe-Corona-Fond and for the support of our SARS-CoV-2 research. M.W. and R.M. were supported by the Deutsche Forschungsgemeinschaft (DFG, WI 5086/1–1, MA 1876/13-1, and MA 1876/12-1). All authors have read and agreed to the published version of the chapter.
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Wilhelm, A., Pallas, C., Marschalek, R., Widera, M. (2022). Detection and Quantification of SARS-CoV-2 by Real-Time RT-PCR Assay. In: Chu, J.J.H., Ahidjo, B.A., Mok, C.K. (eds) SARS-CoV-2. Methods in Molecular Biology, vol 2452. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2111-0_6
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DOI: https://doi.org/10.1007/978-1-0716-2111-0_6
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