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

To test whether direct RT-qPCR could correctly detect the presence (or absence) of SARS-CoV-2 in clinical samples we obtained five aliquots of nasopharyngeal swab samples stored in transport medium at -20°C. Aliquots of the same samples had previously been clinically diagnosed using conventional RNA extraction (MagNA Pure 96 DNA and Viral NA SV Kit, Roche Diagnostics 06543588001) followed by RT-qPCR, calling three patient samples as SARS-CoV-2 positive and two as negative to the virus (Clinical diagnostics performed at the Karolinska University Hospital, Stockholm). We inactivated the nasopharyngeal samples either by adding MagNA Pure 96 External Lysis Buffer (Roche, 06374913001), used in conventional RNA purification, or by heating at 65°C for 30 min, and performed RT-qPCR using 3µl of sample. We observed a lack of amplification in SARS-CoV-2 positive samples inactivated with External Lysis Buffer ( Fig. 2e-f and Supplementary Fig. 1b) . However, RT-qPCR performed directly on heatinactivated samples correctly detected SARS-CoV-2 in all positive samples and lacked signal in the negative samples and controls ( Fig. 2e-f ). This indicated the viability in further exploring heat-inactivated direct RT-qPCR (hid-RT-qPCR) as a method to detect SARS-CoV-2 in clinical samples. We also tested two-step RT and qPCR for SARS-CoV-2 detection on the same clinical samples (Methods), correctly detecting the viral presence and absence (Supplementary Fig. 1c ).