A culture-free method for rapidly and accurately quantifying active SARS-CoV-2

Area of Science:

• Virology
• Molecular Biology
• Infectious Diseases

Background:

• Accurate quantification of infectious virus is crucial for assessing infection risk.
• Traditional median tissue culture infectious dose (TCID50) assays are time-consuming (5-7 days) and require biosafety level 3 laboratories.
• Rapid and sensitive methods are needed for effective management of viral outbreaks like COVID-19.

Purpose of the Study:

• To develop and validate a rapid, culture-free method for quantifying active SARS-CoV-2.
• To target subgenomic RNA (sgRNA) using reverse transcription digital PCR (RT-dPCR) for accurate viral load determination.
• To establish a correlation between sgRNA levels and infectious virus titers.

Main Methods:

• Developed a reverse transcription digital PCR (RT-dPCR) assay targeting SARS-CoV-2 subgenomic RNA (sgRNA-N and sgRNA-E).
• Investigated the dynamic range, limit of detection (LoD = 2 copies/reaction), and limit of quantification (LoQ = 10 copies/reaction) of the RT-dPCR assay.
• Validated the method using cultured SARS-CoV-2 (delta strain) and 63 clinical samples, comparing results with TCID50 assays.

Main Results:

• The RT-dPCR method demonstrated excellent linearity (R² = 0.9999) between sgRNA/gRNA copy numbers and SARS-CoV-2 TCID50 titers (500-10^5 TCID50/mL).
• Quantification of sgRNA-N by RT-dPCR proved more sensitive for active virus detection compared to other methods.
• The developed assay can infer active virus titers within 3 hours in a biosafety level 2 laboratory.

Conclusions:

• RT-dPCR targeting SARS-CoV-2 sgRNA provides a rapid, accurate, and sensitive alternative to traditional TCID50 assays.
• This method enables timely identification of infectious patients, potentially reducing unnecessary isolation periods.
• The assay's efficiency can alleviate the burden on medical resources during widespread COVID-19 outbreaks.