Detection and Quantification of SARS-CoV-2 by Real-Time RT-PCR Assay.
Methods Mol Biol
; 2452: 75-98, 2022.
Article
in English
| MEDLINE | ID: covidwho-1844261
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.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
SARS-CoV-2
/
COVID-19
Type of study:
Diagnostic study
Limits:
Humans
Language:
English
Journal:
Methods Mol Biol
Journal subject:
Molecular Biology
Year:
2022
Document Type:
Article
Affiliation country:
978-1-0716-2111-0_6
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