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

We started by investigating how transport media used for swab collection affect RT-qPCR. To do this, we spiked synthetic full-genome SARS-CoV-2 RNA (SKU102024-MN908947.3, Twist Biosciences) into dilution series of three different transport media (Virocult MED-MW951S, Sigma; Transwab MW176S, Sigma, and Eswab 482C, COPAN) used for clinical sampling at the time of the study (Karolinska University Hospital, Stockholm, Sweden). Each mock sample contained 50,000 synthetic SARS-CoV-2 gRNA copies and 95-0.1% medium, corresponding to 47.5-0.05% medium in the RT-qPCR reaction. We performed single-reaction RT-qPCR using 10µl sample in a 20µl TaqPath reaction and the CDC nucleocapsid 1 (N1) primer-probe set ( Table 1 , Methods) and recorded cycle threshold (C T) values for the dilution series of the media. We observed inhibitory effects in all three media and, importantly, pronounced variation between media (Fig. 2a-d) . Virocult and Transwab demonstrated similar profiles of inhibition, resulting in +2-3 CT at the highest medium concentrations ( Fig. 2a-b) and minimal inhibition at concentrations below 30% medium in the RT-qPCR reaction. Eswab completely impeded detection at high concentrations but reached a similarly low level of inhibition as Virocult and Transwab at 25% concentration in the reaction. For RT experiments relying on synthetic RNA, it is vital to exclude the possibility of lingering DNA template molecules. Therefore, we additionally performed RT and qPCR reactions in two separate steps (Methods) including RT+/-controls, which demonstrated the lack of DNA amplification signal when the reverse transcriptase was eliminated from the reaction (Supplementary Fig. 1a) . Together, these results indicate minimal or no inhibition of Virocult, Transwab and Eswab at ≤25% in the RT-qPCR reaction, corresponding to ≤5µl sample in a 20µl SARS-CoV-2 RT-qPCR reaction.