Department of Medicine, University of California, San Francisco, CA, USA; Department of Medicine, San Francisco VA Health Care System, San Francisco, CA, USA.
Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA.
J Virol Methods. 2021 Jun;292:114115. doi: 10.1016/j.jviromet.2021.114115. Epub 2021 Mar 2.
A hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of sensitive, quantitative RT-ddPCR assays designed to target regions spanning the genome of SARS-CoV-2. Our assays target untranslated regions (5', 3') as well as different coding regions, including non-structural genes that are only found in full length (genomic) RNA and structural genes that are found in genomic as well as different subgenomic RNAs. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 gene expression and may also inform the development of improved diagnostic tools and therapeutics.
冠状病毒转录的一个标志是产生负义 RNA 中间体，作为合成正义基因组 RNA (gRNA) 和一系列亚基因组 mRNA (sgRNA) 的模板，这些 sgRNA 包含来自不连续转录的序列。现有的基于 PCR 的 SARS-CoV-2 诊断检测方法是定性或半定量的，无法提供评估 SARS-CoV-2 在感染过程中复杂转录动态所需的分辨率。我们开发并验证了一组新的敏感、定量 RT-ddPCR 检测方法，旨在针对 SARS-CoV-2 基因组跨越的区域。我们的检测方法针对非翻译区 (5'、3') 以及不同的编码区，包括仅在全长 (基因组) RNA 中发现的非结构基因和在基因组以及不同亚基因组 RNA 中发现的结构基因。将这些检测方法应用于临床相关样本将增强我们对 SARS-CoV-2 基因表达的理解，也可能为开发改进的诊断工具和治疗方法提供信息。
