Validation of the VIRSeek SARS-CoV-2 Mplex Assay for Detection of SARS-CoV-2 on Stainless-Steel Surfaces: AOAC Performance Tested MethodSM 122006.

Abstract

BackgroundThe Eurofins GeneScan Technologies’ VIRSeek SARS-CoV-2 Mplex kit is a RT-qPCR assay for the detection of two targets on the N-gene (nucleocapsid) of SARS-CoV-2. An extraction control, that allows monitoring of the extraction procedure and PCR inhibition, is included.ObjectiveIn-silico analysis and wet testing showed inclusivity and exclusivity of the assay. The complete workflow starting from surface swabbing (VIRSeek PATHOSwab kit), RNA extraction (VIRSeek RNAExtractor), RT-PCR (VIRSeek SARS-CoV-2 Mplex) and evaluation with FastFinder was validated in comparison to the CDC method for detection of SARS-CoV-2 on stainless steel.MethodsIn-silico analysis was performed by using the MFOLD online program. The matrix study was performed for stainless steel inoculated with SARS-CoV-2 isolated from the first documented US case of a traveler from Wuhan, China.ResultsFor inclusivity, 15,764 sequences were analyzed and all mismatches (0.37% of the sequences had single mismatches) were considered non-critical. Cross reactivity for closely related viruses and background organisms was performed, resulting in correct exclusion of all. No significant differences were observed for the POD study when comparing to the CDC method.ConclusionsResults of the inclusivity and exclusivity study show that the assay is specific for detection SARS-CoV-2. The POD study showed no statistically significant difference compared to the CDC reference method, results were identical for the uninoculated and the high level. For the fractional recovery level, the candidate method detected 9/17 samples leading to a POD of 0.47, the reference method detected 11/20 samples leading to a POD of 0.55.