Development of a rapid viability RT-PCR (RV-RT-PCR) method to detect infectious SARS-CoV-2 from swabs

Area of Science:

• Virology
• Molecular Biology
• Environmental Science

Background:

• The COVID-19 pandemic, caused by SARS-CoV-2, necessitates rapid detection methods for surface transmission and environmental epidemiology.
• Current gold-standard methods for detecting infectious SARS-CoV-2, such as cell-culture-based assays, require several days for results.
• Faster detection of viable SARS-CoV-2 is crucial for public health interventions and research.

Purpose of the Study:

• To develop and validate a rapid viability reverse transcriptase PCR (RV-RT-PCR) method for detecting infectious SARS-CoV-2 from swab samples.
• To significantly reduce the time required for detecting viable SARS-CoV-2 compared to traditional cell-culture methods.
• To provide a tool for environmental epidemiological investigations and studies on virus surface transmission.

Main Methods:

• Developed an RV-RT-PCR method integrating cell-culture-based viral enrichment in a 96-well plate format.
• Utilized gene-specific RT-PCR analysis before and after sample incubation to determine the cycle threshold difference (ΔC_T).
• Incorporated ultrafiltration for virion concentration and an algorithm (ΔC_T ≥ 6) to identify infectious virus based on RNA increase.

Main Results:

• The RV-RT-PCR method detects viable SARS-CoV-2 in <1 day (17 hours for a batch of 12 swabs).
• Achieved detection of <50 SARS-CoV-2 virions in swab samples.
• Demonstrated a significant time reduction compared to the days required by cell-culture-based methods.

Conclusions:

• The developed RV-RT-PCR method offers a rapid and sensitive approach for detecting infectious SARS-CoV-2.
• This method can expedite environmental and epidemiological studies of SARS-CoV-2 transmission.
• The RV-RT-PCR approach may be applicable for clinical sample analysis, antiviral drug testing, and developing rapid methods for other viruses.