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Development of Rapid Viability-Reverse Transcriptase PCR (RV-RTPCR) Method for Detection of Infectious SARS-CoV-2 from Environmental Samples
Citation:
Shah, S., S. Kane, M. Elsheikh, AND T. Alfaro. Development of Rapid Viability-Reverse Transcriptase PCR (RV-RTPCR) Method for Detection of Infectious SARS-CoV-2 from Environmental Samples. U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-21/059, 2021.
Impact/Purpose:
At the onset of the COVID-19 pandemic, the EPA-ORD’s Homeland Security Research Program expanded their research horizons to respond to this public health emergency. Surface contamination, stability of the COVID-19 causing virus (SARS-CoV-2) on different surfaces, and potential indirect transmission of the virus via surfaces have been reported by many researchers. However, surface transmission of SARS-CoV-2 is still not well understood, and its overall importance in viral spread is mostly unknown. A rapid analytical method for detection of infectious (live) SARS-CoV-2 in environmental surface samples (e.g., swabs) is needed to better understand the surface transmission of this virus and to support environmental epidemiological investigations. To address this need, a proof-of-concept Rapid Viability-Reverse Transcriptase Polymerase Chain Reaction (RV-RTPCR) method was developed. This report describes the method development research. The RV-RTPCR method will allow detection of infectious SARS-CoV-2 in environmental surfaces and any other sample types within hours, rather than several days taken by current cell-culture-based methods. The ability to rapidly detect infectious SARS-CoV-2 in environmental samples will be valuable for epidemiology investigations and environmental surveillance in healthcare facilities, within a facility (e.g., prison, nursing home), between facilities, and within a community.
Description:
This report describes the development of a Rapid Viability-Reverse Transcriptase PCR (RV-RTPCR) method for detection of infectious (live) virus (SARS-CoV-2) that causes COVID-19. The EPA-ORD’s Homeland Security Research Program (HSRP), in collaboration with the Department of Energy’s Lawrence Livermore National Laboratory (LLNL), expeditiously developed this method to detect infectious SARS-CoV-2 in environmental surfaces samples in hours, rather than several days typical of current cell-culture-based methods. Basically, the RV-RTPCR method integrates cell-culture based enrichment of the virus in a sample with virus-gene-specific RTPCR-based molecular analysis. The RTPCR analysis is conducted before and after virus (sample) incubation in cell-culture. An algorithm based on the cycle threshold (CT) difference (¿CT) of 6 or greater, representing approximately 2-log or higher increase in viral RNA content, between before and after cell-culture-virus incubation RTPCR analyses determines the presence of infectious virus in the sample.