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

Autor: Telwatte S; 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., Martin HA; 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., Marczak R; University of California, Santa Barbara, CA, United States., Fozouni P; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, United States., Vallejo-Gracia A; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, United States., Kumar GR; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, United States., Murray V; Department of Medicine, University of California, San Francisco (UCSF), San Francisco, CA, United States., Lee S; Department of Medicine, University of California, San Francisco (UCSF), San Francisco, CA, United States., Ott M; 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., Wong JK; 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., Yukl SA; 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: Steven.yukl@ucsf.edu.
Jazyk: angličtina
Zdroj: Methods (San Diego, Calif.) [Methods] 2022 May; Vol. 201, pp. 15-25. Date of Electronic Publication: 2021 Apr 18.
DOI: 10.1016/j.ymeth.2021.04.011
Abstrakt: 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.)
Databáze: MEDLINE
Externí odkaz: