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

Authors
  • Telwatte, Sushama1
  • Martin, Holly Anne1
  • Marczak, Ryan2
  • Fozouni, Parinaz3
  • Vallejo-Gracia, Albert3
  • Kumar, G Renuka3
  • Murray, Victoria4
  • Lee, Sulggi4
  • Ott, Melanie5
  • Wong, Joseph K1
  • Yukl, Steven A6
  • 1 Department of Medicine, University of California, San Francisco (UCSF), San Francisco, CA, United States; Department of Medicine, San Francisco VA Health Care System, San Francisco, CA, United States. , (United States)
  • 2 University of California, Santa Barbara, CA, United States. , (United States)
  • 3 Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, United States. , (United States)
  • 4 Department of Medicine, University of California, San Francisco (UCSF), San Francisco, CA, United States. , (United States)
  • 5 Department of Medicine, University of California, San Francisco (UCSF), San Francisco, CA, United States; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, United States. , (United States)
  • 6 Department of Medicine, University of California, San Francisco (UCSF), San Francisco, CA, United States; Department of Medicine, San Francisco VA Health Care System, San Francisco, CA, United States. Electronic address: [email protected] , (United States)
Type
Published Article
Journal
Methods
Publisher
Elsevier
Publication Date
May 01, 2022
Volume
201
Pages
15–25
Identifiers
DOI: 10.1016/j.ymeth.2021.04.011
PMID: 33882362
Source
Medline
Keywords
Language
English
License
Unknown

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. Published by Elsevier Inc.

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