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

One-step RT-qPCR: For reverse transcription and quantitative PCR we used the one-step TaqPath RT-qPCR master mix (Thermo, A15299) according to manufacturer's instructions. Total reactions of 20µl were formed by mixing 5µl TaqPath master mix, primers-probe mix, sample and RNase free water to fill the reaction. Primer and probe concentrations in the RT-qPCR reaction were as follows, E and RdRP: 300nM each primer, 200nM probe; N1 and RdRP: 500nM each primer, 125nM probe. Primer and probe sequences are listed in Table 1 . The thermal cycling steps were: 25 o C for 2 min, 50 o C for 15min, 95 o C for 2min, and 45 cycles of 95 o C for 3s and 56 o C for 30s. RT-qPCR was performed on a Step-One-Plus real time PCR machine (Applied Biosystems) using the StepOne Software v2.3. The samples from the self-test screen (Fig. 4e) were subjected to the same protocol as describe above, but without heat inactivation. Briefly, samples (swab samples in PBS or pure saliva) were mixed with equal volume of 10% or 20% Triton X-100 at room temperature (approximately 5min) before dilution 1:1 in a 10µl TaqPath RT-qPCR master mix as described above. Clinical COVID-19 diagnostics at the Karolinska University Hospital, Stockholm, Sweden were similarly performed using TaqPath and primer and probes for E and RdRP and 10µl RNA eluate as sample.