Development of Rapid Viability-Reverse Transcriptase PCR (RV-RTPCR) Method for Detection of Viable SARS-CoV-2 from Environmental Samples
U.S. Environmental Protection Agency (EPA) scientists have expanded their research horizons in the wake of the current COVID-19 public health emergency caused by SARS-CoV-2. Several research projects are underway to pave the way for better understanding and reducing the risk of exposure to SARS-CoV-2. This research will help states and territories, tribes, and local governments—including public health agencies—and guide business owners, homeowners, and others in reducing the risk of exposure, which in turn will save lives.
Surface transmission of SARS-CoV-2 is not well understood, and its overall importance in COVID-19 transmission is not well known. Both asymptomatic and pre-symptomatic carriers of SARS-CoV-2 can shed the virus and contaminate surfaces,. Coronaviruses can survive and remain infectious on some inanimate surfaces at room temperature (below 30ºC) for days. Because it takes several days to obtain results from currently available cell-culture-based methods that can detect live/viable (infectious) SARS-CoV-2, it is difficult to quickly assess the virus survival period on real-world surfaces and understand surface transmission. These time-consuming methods seriously impact environmental epidemiology investigations and transmission studies, where timely knowledge of the presence of infectious virus on a surface is critical. Therefore, a rapid, dependable and accurate analytical method for detecting live/viable SARS-CoV-2 in environmental surface samples (e.g., swabs) is needed to expedite understanding surface transmission of this virus. The ability to rapidly detect live/infectious SARS-CoV-2 would be valuable for epidemiology investigations in healthcare facilities, within a facility (e.g., prison, nursing home), between facilities, and within a community. The purpose of this research effort is to develop a Rapid Viability-Reverse Transcriptase PCR (RV-RTPCR) method that will allow detection of live SARS-CoV-2 in one day, rather than several days typical of the currently used cell-culture-based methods.
 Arons et al. N Engl J Med 2020; 382:2081-2090. DOI: 10.1056/NEJMoa2008457
 Kampf et al. J Hosp Infect 104 (2020) 246e251
The most commonly used analytical methods [immunoassays and reverse transcriptase polymerase chain reaction (RTPCR) assays] can detect the SARS-CoV-2 in a sample via a specific protein on the virus surface or a gene in its genomic material (RNA), respectively. However, the results of these rapid methods do not indicate whether the live virus is present in the sample. To detect live/viable SARS-CoV-2 in a sample, a traditional cell-culture-based analytical method must be used. This traditional culture method can take several days to provide definitive results on the presence or absence of live/viable virus in a sample. There is an urgent need for an alternative and rapid analytical method that can detect live/viable SARS-CoV-2 in a relatively short time to improve the ability to understand surface transmission.
The objective of this research is the expeditious development of a rapid and sensitive analytical method that can detect live/viable SARS-CoV-2 with molecular (RTPCR) confirmation in a significantly shorter time than the traditional cell-culture based method. The development of this method is building on the EPA success in developing rapid viability PCR (RV-PCR) methods for high-priority biothreat agents such as Bacillus anthracis (bacteria causing anthrax), Yersinia pestis (bacteria causing plague), and Francisella tularensis (bacteria causing tularemia). This new method for SARS-CoV-2 viability testing is designated as Rapid Viability - Reverse Transcriptase Polymerase Chain Reaction (RV-RTPCR). Since all research directly with the SARS-CoV-2 strain requires working in a Biosafety Level-3 (BSL-3) laboratory, a small laboratory footprint approach for the new method will be adopted, resulting in the generation of less BSL-3 laboratory waste than the traditional method. The small footprint and reduced waste primarily results from (1) significantly fewer multi-well plates compared to cell-culture plates required for traditional methods, (2) lower volumes of growth medium and reagents, and (3) less solid waste due to use of micropipet tips for small volumes rather than large serological pipets required with the use of traditional methods.
Under the EPA’s technical direction and with input from the project team that includes subject matter experts from the Centers for Disease Control and Prevention (CDC), the SARS-CoV-2 RV-RTPCR method development research is being conducted in collaboration with scientists at the Department of Energy’s Lawrence Livermore National Laboratory.
EPA is conducting several research projects to inform the response to the COVID pandemic caused public health emergency. The interim results summary here is intended to provide a simple representation of the results of an on-going method development research effort; therefore, only a brief description of the purpose of the effort, methods, and interim results are provided. This research effort is being conducted in accordance with an approved Quality Assurance Project Plan. The interim results have been reviewed by internal EPA technical experts, quality assurance staff, and management. No interpretation of the interim results is provided. Once complete, the study and its results will be described in detail in a publication subjected to external, expert peer review. EPA does not endorse the purchase or sale of any commercial products or services.