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.
University of California, Santa Barbara, CA, United States.
Methods. 2022 May;201:15-25. doi: 10.1016/j.ymeth.2021.04.011. Epub 2021 Apr 18.
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.
SARS-CoV-2 和其他冠状病毒的复制依赖于负义 RNA 中间体的转录,这些中间体作为合成正链基因组 RNA (gRNA) 和多个不同亚基因组 mRNA (sgRNA) 的模板,其中包含来自不连续转录的片段。最近的研究旨在描述亚基因组 SARS-CoV-2 转录本的表达,以研究其临床意义。在这里,我们描述了一组新的逆转录液滴数字 PCR (RT-ddPCR) 检测,旨在专门定量多个不同的亚基因组 SARS-CoV-2 转录本,并将其与每个基因座不通过不连续转录产生的转录本区分开来。这些检测可以应用于感染 SARS-CoV-2 的患者样本,以更好地了解 SARS-CoV-2 转录的调控以及不同的 sgRNA 如何导致病毒发病机制和临床疾病严重程度。