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通过数字液滴 PCR 和元转录组学分析准确检测和定量 SARS-CoV-2 基因组和亚基因组 mRNA。

Accurate detection and quantification of SARS-CoV-2 genomic and subgenomic mRNAs by ddPCR and meta-transcriptomics analysis.

机构信息

Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari Aldo Moro, Via Orabona 4, 70126, Bari, Italy.

Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies, National Research Council, Via Amendola 122/O, 70126, Bari, Italy.

出版信息

Commun Biol. 2021 Oct 22;4(1):1215. doi: 10.1038/s42003-021-02748-0.

Abstract

SARS-CoV-2 replication requires the synthesis of a set of structural proteins expressed through discontinuous transcription of ten subgenomic mRNAs (sgmRNAs). Here, we have fine-tuned droplet digital PCR (ddPCR) assays to accurately detect and quantify SARS-CoV-2 genomic ORF1ab and sgmRNAs for the nucleocapsid (N) and spike (S) proteins. We analyzed 166 RNA samples from anonymized SARS-CoV-2 positive subjects and we observed a recurrent and characteristic pattern of sgmRNAs expression in relation to the total viral RNA content. Additionally, expression profiles of sgmRNAs, as determined by meta-transcriptomics sequencing of a subset of 110 RNA samples, were highly correlated with those obtained by ddPCR. By providing a comprehensive and dynamic snapshot of the levels of SARS-CoV-2 sgmRNAs in infected individuals, our results may contribute a better understanding of the dynamics of transcription and expression of the genome of SARS-CoV-2 and facilitate the development of more accurate molecular diagnostic tools for the stratification of COVID-19 patients.

摘要

SARS-CoV-2 的复制需要通过十个亚基因组 mRNA(sgmRNAs)的不连续转录来合成一组结构蛋白。在这里,我们对液滴数字 PCR(ddPCR)检测进行了微调,以准确检测和定量 SARS-CoV-2 基因组 ORF1ab 和核衣壳(N)和刺突(S)蛋白的 sgmRNAs。我们分析了来自匿名 SARS-CoV-2 阳性受试者的 166 个 RNA 样本,观察到 sgmRNAs 的表达与总病毒 RNA 含量之间存在反复出现的特征性模式。此外,通过对 110 个 RNA 样本的亚转录组学测序确定的 sgmRNAs 表达谱与 ddPCR 获得的表达谱高度相关。通过提供受感染个体中 SARS-CoV-2 sgmRNAs 水平的全面和动态快照,我们的结果可能有助于更好地理解 SARS-CoV-2 基因组转录和表达的动态,并促进开发更准确的分子诊断工具,以对 COVID-19 患者进行分层。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4403/8536764/3954352f7e2e/42003_2021_2748_Fig1_HTML.jpg

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