tetano
Editor, Senior Moderator
Methods
. 2021 Apr 18;S1046-2023(21)00103-1.
doi: 10.1016/j.ymeth.2021.04.011. Online ahead of print.
Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts
Sushama Telwatte[SUP] 1 [/SUP], Holly Anne Martin[SUP] 1 [/SUP], Ryan Marczak[SUP] 2 [/SUP], Parinaz Fozouni[SUP] 3 [/SUP], Albert Vallejo-Gracia[SUP] 3 [/SUP], G Renuka Kumar[SUP] 3 [/SUP], Victoria Murray[SUP] 4 [/SUP], Sulggi Lee[SUP] 4 [/SUP], Melanie Ott[SUP] 5 [/SUP], Joseph K Wong[SUP] 1 [/SUP], Steven A Yukl[SUP] 6 [/SUP]
Affiliations
Abstract
The replication of SARS-CoV-2 and other coronaviruses depends on transcription of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and multiple different subgenomic mRNAs (sgRNAs) encompassing fragments arising from discontinuous transcription. Recent studies have aimed to characterize the expression of subgenomic SARS-CoV-2 transcripts in order to investigate their clinical significance. Here, we describe a novel panel of reverse transcription droplet digital PCR (RT-ddPCR) assays designed to specifically quantify multiple different subgenomic SARS-CoV-2 transcripts and distinguish them from transcripts that do not arise from discontinuous transcription at each locus. These assays can be applied to samples from SARS-CoV-2 infected patients to better understand the regulation of SARS-CoV-2 transcription and how different sgRNAs may contribute to viral pathogenesis and clinical disease severity.
Keywords: COVID-19; Coronavirus; Digital PCR; Droplet digital PCR; Quantitative assays; SARS-CoV-2; Subgenomic RNA; Viral transcription/replication.
. 2021 Apr 18;S1046-2023(21)00103-1.
doi: 10.1016/j.ymeth.2021.04.011. Online ahead of print.
Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts
Sushama Telwatte[SUP] 1 [/SUP], Holly Anne Martin[SUP] 1 [/SUP], Ryan Marczak[SUP] 2 [/SUP], Parinaz Fozouni[SUP] 3 [/SUP], Albert Vallejo-Gracia[SUP] 3 [/SUP], G Renuka Kumar[SUP] 3 [/SUP], Victoria Murray[SUP] 4 [/SUP], Sulggi Lee[SUP] 4 [/SUP], Melanie Ott[SUP] 5 [/SUP], Joseph K Wong[SUP] 1 [/SUP], Steven A Yukl[SUP] 6 [/SUP]
Affiliations
- PMID: 33882362
- DOI: 10.1016/j.ymeth.2021.04.011
Abstract
The replication of SARS-CoV-2 and other coronaviruses depends on transcription of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and multiple different subgenomic mRNAs (sgRNAs) encompassing fragments arising from discontinuous transcription. Recent studies have aimed to characterize the expression of subgenomic SARS-CoV-2 transcripts in order to investigate their clinical significance. Here, we describe a novel panel of reverse transcription droplet digital PCR (RT-ddPCR) assays designed to specifically quantify multiple different subgenomic SARS-CoV-2 transcripts and distinguish them from transcripts that do not arise from discontinuous transcription at each locus. These assays can be applied to samples from SARS-CoV-2 infected patients to better understand the regulation of SARS-CoV-2 transcription and how different sgRNAs may contribute to viral pathogenesis and clinical disease severity.
Keywords: COVID-19; Coronavirus; Digital PCR; Droplet digital PCR; Quantitative assays; SARS-CoV-2; Subgenomic RNA; Viral transcription/replication.