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

ABSTRACT

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.