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

Area of Science:

• Virology
• Immunology
• Molecular Biology

Background:

• Persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA/protein/virus is detectable months post-infection.
• This persistence may be linked to SARS-CoV-2 variants or long coronavirus disease (Long COVID).
• Detecting low viral loads is crucial for understanding persistent infection and Long COVID.

Purpose of the Study:

• To develop and present a protocol for detecting low levels of SARS-CoV-2 RNA and protein simultaneously.
• To enable sensitive quantification and visualization of persistent viral presence in clinical samples.
• To provide a method applicable to research on SARS-CoV-2 variants and Long COVID.

Main Methods:

• Protocol involves cell infection with SARS-CoV-2.
• Quantification using fluorescence in situ hybridization-flow cytometry (FISH-flow cytometry).
• Visualization via immunolabeling and RNAscope techniques.

Main Results:

• The described protocol effectively detects low levels of viral RNA and protein.
• FISH-flow cytometry allows for sensitive quantification.
• Immunolabeling and RNAscope provide detailed visualization of viral components.

Conclusions:

• The presented protocol offers a sensitive approach for detecting persistent SARS-CoV-2.
• This method is directly applicable to clinical samples for Long COVID research.
• The technique aids in understanding the mechanisms of prolonged viral presence and its implications.