A comparative study of real-time RT-PCR based SARS-CoV-2 detection methods and its application to human derived and surface swabbed material

ABSTRACT

Real-time reverse transcription polymerase chain reaction (RT-PCR) remains a gold standard in detection of various viral diseases. In the COVID-19 pandemic, multiple RT-PCR based tests were developed to screen for viral infection. As an emergency response to growing testing demand, we established a SARS-CoV-2 PCR diagnostics platform for which we compared different commercial and in-house RT-PCR protocols. We evaluated four commercial (CDC 2019-nCoV, Applied Biosystems 2019-nCoV Assay Kit v1 TF-SinglePlex, 2019-nCoV Assay Kit v2 TF-MultiPlex, and EURORealTime SARS-CoV-2), one customized (Institute Pasteur), and one in-house RT-PCR protocols with 92 SARS-CoV-2 positive and 92 SARS-CoV-2 negative samples. Furthermore, we compared economical and practical characteristics of these protocols. We also developed a highly sensitive digital droplet PCR (ddPCR) method. Finally, we conducted a local environmental study for the presence and infectivity of SARS-CoV-2 on different surfaces in a quarantined household using RT- and ddPCR methods. We found very low limits of detection (1 or 2 viral copies/L), high sensitivities (93.6-97.8%) and specificities (98.7-100%) for the tested RT-PCR protocols. We further demonstrated the feasibility of downscaling two of the commercial protocols, which could optimize testing capacity. In the local environmental study, only one surface sample tested positive for viral RNA, but without detectable infectivity in vitro. Tested commercial and customized RT-PCR detection kits show very good and comparable sensitivity, and specificity, and the kits could be further optimized for use on SARS-CoV-2 viral samples derived from human and surface swabbed samples.