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

Two-step RT-qPCR: Reverse transcription was performed by mixing 1-3 µl subject sample, 1µl 10mM dNTPs, 0.15µl 50µM random hexamers (N8080127, Thermo), 0.1µl RNase inhibitor (2313B, TaKaRa), 0.4µl 0.5% Triton X-100 and RNase free water up to 4.5µl, followed by incubation at 72 o C for 3min. The sample was placed on ice and a mix containing 0.5µl 100mM DTT, 2µl 5M betaine, 0.1µl 1M MgCl2, 0.25µl RNase inhibitor (2313B, TaKaRa), 2µl 5x Superscript II buffer, 0.5µl Superscript II (Invitrogen) and water up to 5.5µl was added. The samples were then incubated at 25 o C for 10min followed by 42 o C for 25min and finally for 70 o C for 15min. Amplification of 10µl cDNA (RT mix) using primers and probes described in Table 1 was performed using BioTaq DNA polymerase (Bio-21040, Bioline) in a 20µl reaction containing 2µl 10x NH4 Reaction Buffer (Bioline), 1.2µl 50mM MgCl2 , 0.2µl 100mM dNTP Mix and water up to 20 µl. The thermal cycling steps were: 25 o C for 2min, 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. qPCR was performed on a Step-One-Plus real time PCR machine (Applied Biosystems) using the StepOne Software v2.3.