Comparison of the Modified Centers for Disease Control and Prevention 2019-Novel Coronavirus Real-Time RT-PCR Method for Detection of Infectious and Heat-Inactivated Virus on Stainless Steel

Brunelle, Sharon L; Bird, Patrick M; Boone, Jeremy; Nelson, Maria; Johnson, Zerlinde; Coates, Scott

BACKGROUND: Infectious Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was used in the validation of methods for detection of SARS-CoV-2 on stainless steel surfaces in the AOAC Research Institute Emergency Response Validation project. Handling infectious virus requires Biosafety Level (BSL)-3 facilities. OBJECTIVE: To compare the recovery and detection of infectious and heat-inactivated (65 °C for 30 min) SARS-CoV-2 from stainless steel by the modified US Centers for Disease Control and Prevention (CDC) 2019-Novel Coronavirus Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Diagnostic Panel. METHODS: Viral stocks were diluted in viral transport medium and deposited onto stainless steel test areas at 2 x 10(3) and 2 x 10(4) genomic copies for low and high, respectively. Test areas were sampled, and aliquots of the resulting test solutions analyzed by RT-qPCR according to the CDC method. Results were analyzed by Probability of Detection (POD) statistics. RESULTS: The low level, where fractional positive results (25–75%) are expected, yielded POD(I) = 0.80 (0.58, 0.92) for the infectious virus and POD(HI) = 0.15 (0.05, 0.36) for the heat-inactivated virus. The bias, dPOD(HI) = -0.65 (-0.80, -0.35), demonstrated a statistical difference between infectious and heat-inactivated virus detection. No difference was observed at the high inoculation level. CONCLUSION: Despite the statistical difference observed, the use of the heat-inactivated virus is a viable alternative for matrix extension studies using a method comparison study design. HIGHLIGHTS: The use of heat-inactivated SARS-CoV-2 can mitigate the need for a BSL-3 facility for matrix extension validation of alternative methods in SARS-CoV-2 studies.