Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-qPCR

Ioanna Smyrlaki; Martin Ekman; Martin Vondracek; Natali Papanicoloau; Antonio Lentini; Johan Aarum; Shaman Muradrasoli; Jan Albert; Björn Högberg; Björn Reinius

Next, we tested primer-probe set performance in hid-RT-qPCR using nasopharyngeal swab samples and primers-probe sets targeting the SARS-CoV-2 genes RNA-dependent RNA polymerase (RdRP), envelope (E), and nucleocapsid (N1) ( Table 1) . We obtained additional heat-inactivated (65°C, 30 min) nasopharyngeal swab samples called as SARS-CoV-2 positive in previous clinical diagnostics (Methods). We observed, in our setting, a modest difference between N1 and RdRP in hid-RT-qPCR (mean and median CT difference to N1: 0.63 and 0.27, P=0.032, Wilcoxon signed-rank test) while the E-gene set appeared at considerably higher CT values than the other primerprobe sets (mean and median CT difference to N1: 2.9 and 1.7, P=0.00098, Wilcoxon signed-rank test) ( Fig. 2g-h) . Lower performance of primers and probes targeting the E gene is in line with previous results [7, 11] . We argue that short amplicon targets are optimal for heat-inactivated samples, while considerations on the amplicon length could be less important for PCR amplification performed on extracted RNA from fresh samples (See Discussion). Due to the superior performance of N1 and RdRP in hid-RT-qPCR we focused on these primers in all further analyses.