Protocol to detect infectious SARS-CoV-2 at low levels using in situ hybridization techniques

Low and persistent levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA/protein/virus can be detected in clinical samples months after infection, possibly related to the emergence of SARS-CoV-2 variants or development of long coronavirus disease. Here, we present a protocol to detect low levels of viral RNA together with protein using flow cytometry and microscopy. We describe steps for cell infection with SARS-CoV-2 and quantification by fluorescence in situ hybridization-flow cytometry. We then detail procedures for visualization using immunolabeling and RNAscope. This approach is directly applicable to clinical samples. For complete details on the use and execution of this protocol, please refer to Zhu et al. (2022).1 [Display omitted] •Protocol to quantify low level of infectious SARS-CoV-2 at the single-cell level•Immunolabeling and in situ hybridization details to detect spike protein and viral RNA•Combining morphological and flow cytometry quantifications for improving robustness•Guidance to deal with background and to improve signal-to-noise ratios Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Low and persistent levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA/protein/virus can be detected in clinical samples months after infection, possibly related to the emergence of SARS-CoV-2 variants or development of long coronavirus disease. Here, we present a protocol to detect low levels of viral RNA together with protein using flow cytometry and microscopy. We describe steps for cell infection with SARS-CoV-2 and quantification by fluorescence in situ hybridization-flow cytometry. We then detail procedures for visualization using immunolabeling and RNAscope. This approach is directly applicable to clinical samples.