Development of a rapid viability RT-PCR (RV-RT-PCR) method to detect infectious SARS-CoV-2 from swabs
Since the rapid onset of the COVID-19 pandemic, its causative virus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), continues to spread and increase the number of fatalities. To expedite studies on understanding potential surface transmission of the virus and to aid environmental epidemiological investigations, here we developed a rapid viability reverse transcriptase PCR (RV-RT-PCR) method that detects viable (infectious) SARS-CoV-2 from swab samples in <1 day compared to several days required by current gold-standard cell-culture-based methods. The method integrates cell-culture-based viral enrichment in a 96-well plate format with gene-specific RT-PCR-based analysis before and after sample incubation to determine the cycle threshold (CT) difference (ΔCT). An algorithm based on ΔCT ≥ 6 representing ~ 2-log or more increase in SARS-CoV-2 RNA following enrichment determines the presence of infectious virus. The RV-RT-PCR method with 2-hr viral infection and 9-hr post-infection incubation periods includes ultrafiltration to concentrate virions, resulting in detection of <50 SARS-CoV-2 virions in swab samples in 17 h (for a batch of 12 swabs), compared to days typically required by the cell-culture-based method. The SARS-CoV-2 RV-RT-PCR method may also be useful in clinical sample analysis and antiviral drug testing, and could serve as a model for developing rapid methods for other viruses of concern.
- Research Organization:
- Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
- Sponsoring Organization:
- USDOE National Nuclear Security Administration (NNSA); USEPA
- Grant/Contract Number:
- AC52-07NA27344; DW-089-92527601-0; EPA IA DW-089-92527601 - 0
- OSTI ID:
- 1862742
- Alternate ID(s):
- OSTI ID: 1862459
- Report Number(s):
- LLNL-JRNL-826285; 1040270
- Journal Information:
- Journal of Virological Methods, Vol. 297; ISSN 0166-0934
- Publisher:
- ElsevierCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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