Detection and Quantification of SARS-CoV-2 by Real-Time RT-PCR Assay.
COVID-19
RdRP
Real time polymerase chain reaction
Reverse transcription
SARS-CoV-2
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
13
5
2022
pubmed:
14
5
2022
medline:
18
5
2022
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 35554902
doi: 10.1007/978-1-0716-2111-0_6
doi:
Substances chimiques
RNA, Viral
0
RNA-Dependent RNA Polymerase
EC 2.7.7.48
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
75-98Informations de copyright
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Références
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