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利用 CoronaFISH 技术对 SARS-CoV-2 RNA 进行灵敏可视化检测。

Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH.

机构信息

Institut Pasteur, Université de Paris, CNRS UMR 3691, Imaging and Modeling Unit, Paris, France.

Institut Pasteur, Université de Paris, CNRS UMR 3569, Unité de Virologie Structurale, Paris, France.

出版信息

Life Sci Alliance. 2022 Jan 7;5(4). doi: 10.26508/lsa.202101124. Print 2022 Apr.

Abstract

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.

摘要

当前的 COVID-19 大流行是由严重急性呼吸系统综合征冠状病毒 2(SARS-CoV-2)引起的。这种正链单链 RNA 病毒包含一个单一线性 RNA 片段,作为转录和复制的模板,导致感染细胞中产生正链和负链病毒 RNA(vRNA)。用于直接在感染细胞中可视化 vRNA 的工具对于分析病毒复制周期、筛选治疗分子或研究人体组织中的感染至关重要。在这里,我们报告了用于可视化 SARS-CoV-2(CoronaFISH)正链或负链 RNA 的 FISH 探针的设计、验证和初步应用。我们证明了在非洲绿猴和几种人类细胞系、患者样本和人体组织中对 vRNA 的敏感可视化。我们进一步证明了 CoronaFISH 探针可适应电子显微镜。我们提供了所有所需的寡核苷酸序列、探针设计的源代码和详细的方案。我们希望 CoronaFISH 将补充现有的 SARS-CoV-2 生物学和 COVID-19 病理生理学研究、药物筛选和诊断技术。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/94ae/8742873/cbbd59a53300/LSA-2021-01124_Fig1.jpg

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