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Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts.

  • Author(s): Telwatte, Sushama;
  • Martin, Holly Anne;
  • Marczak, Ryan;
  • Fozouni, Parinaz;
  • Vallejo-Gracia, Albert;
  • Kumar, G Renuka;
  • Murray, Victoria;
  • Lee, Sulggi;
  • Ott, Melanie;
  • Wong, Joseph K;
  • Yukl, Steven A
  • et al.
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.

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