Development and validation of a new triplex real-time quantitative reverse Transcriptase-PCR assay for the clinical detection of SARS-CoV-2

Gadkar, Vijay; Goldfarb, David M.; Young, Virginia A.; Watson, Nicole; Al‐Rawahi, Ghada N.; Srigley, Jocelyn A.; Tilley, Peter

doi:10.1016/j.mcp.2021.101744

Cited by 13 publications

(15 citation statements)

References 29 publications

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“…Moreover, a quadruplex RT-qPCR assay has been used for the detection of gN and gE along with two human cellular genes [ 23 ]. Some triplex RT-qPCR assays also target the genomic S ( gS ) [ 7 ] and gE or S and ORF8 genes simultaneously with human cellular genes [ 24 , 25 ]. Additionally, some multiplex RT-qPCR assays simultaneously detect two different nidovirals, porcine torovirus (PToV) and coronavirid [ 26 ].…”

Section: Introductionmentioning

confidence: 99%

Duplex One-Step RT-qPCR Assays for Simultaneous Detection of Genomic and Subgenomic RNAs of SARS-CoV-2 Variants

Bhosle

Tran

Yú

et al. 2022

Viruses

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A hallmark of severe acute respiratory syndrome virus (SARS-CoV-2) replication is the discontinuous transcription of open reading frames (ORFs) encoding structural virus proteins. Real-time reverse transcription PCR (RT-qPCR) assays in previous publications used either single or multiplex assays for SARS-CoV-2 genomic RNA detection and a singleplex approach for subgenomic RNA detection. Although multiplex approaches often target multiple genomic RNA segments, an assay that concurrently detects genomic and subgenomic targets has been lacking. To bridge this gap, we developed two duplex one-step RT-qPCR assays that detect SARS-CoV-2 genomic ORF1a and either subgenomic spike or subgenomic ORF3a RNAs. All primers and probes for our assays were designed to bind to variants of SARS-CoV-2. In this study, our assays successfully detected SARS-CoV-2 Washington strain and delta variant isolates at various time points during the course of live virus infection in vitro. The ability to quantify subgenomic SARS-CoV-2 RNA is important, as it may indicate the presence of active replication, particularly in samples collected longitudinally. Furthermore, specific detection of genomic and subgenomic RNAs simultaneously in a single reaction increases assay efficiency, potentially leading to expedited lucidity about viral replication and pathogenesis of any variant of SARS-CoV-2.

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Section: Introductionmentioning

confidence: 99%

Duplex One-Step RT-qPCR Assays for Simultaneous Detection of Genomic and Subgenomic RNAs of SARS-CoV-2 Variants

Bhosle

Tran

Yú

et al. 2022

Viruses

View full text Add to dashboard Cite

show abstract

“… Abbreviations: F, forward; PCR, polymerase chain reaction; R, reverse. a Primers retrieved from Gadkar et al 18 and Qasem et al, 19 and further details and references therein. b Gene names given according to Uniprot.…”

Section: Introductionmentioning

confidence: 99%

“… a Primers retrieved from Gadkar et al 18 and Qasem et al, 19 and further details and references therein. …”

Section: Introductionmentioning

confidence: 99%

“… a Twelve primers described in Table 1 and derived from [ 18 19 ] were analyzed for sharing of nucleotide sequences with the human genome. BLASTn [ 16 17 ] was used to find and localize regions of identity in the human nucleotide collection covering genomic and transcript sequences; further details are available at .…”

Section: Introductionmentioning

confidence: 99%

“…De facto, using the nucleotide Basic Local Alignment Search Tool (BLASTn) program from NCBI (http://blast.ncbi. nlm.nih.gov, 16,17 a sample of 12 primers retrieved from literature, 18,19 proposed/used even by government health institutions 19 to detect SARS-CoV-2, and described here in ►Table 1, was analyzed for nucleotide sequence sharing with the human genome. BLASTn analyses documented a relevant viral versus human oligonucleotide overlap, with shared primer sequences repeatedly present in the human genome, disseminated among different chromosomes, and located in plus strands, minus strands, mRNAs, pseudogenes, etc.…”

Section: Introductionmentioning

confidence: 99%

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