We design an RNA-FISH method to target the plus and minus strands of SARS-CoV-2 RNA, and apply it to image the virus in cell lines, lung tissue, and nasal swabs, by fluorescence and electron microscopy. 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.
【초록키워드】 COVID-19, SARS-CoV-2, coronavirus, protocol, COVID-19 pandemic, Transcription, Infection, complement, Drug screening, severe acute respiratory syndrome Coronavirus, virus, diagnostics, RNA, Replication, electron microscopy, Viral, Screen, pathophysiology, viral replication, Microscopy, Research, Patient, SARS-CoV-2 RNA, Viral RNA, RNA virus, adaptation, respiratory, synthesis, African green monkey, Critical, human cell lines, cell lines, source code, green monkey, acute respiratory syndrome, Template, acute respiratory syndrome coronavirus, tissue, acute respiratory syndrome coronavirus 2, infected cells, lung tissue, vRNA, SARS-CoV-2 biology, probes, positive, nasal swabs, infected cell, therapeutic molecules, probe, oligonucleotide sequences, initial, caused, required, linear, positive-sense single-stranded RNA, 【제목키워드】 sensitive,