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

In blind testing of 85 clinical samples, we found that hid-RT-qPCR and the CDC N1 primer-probe-set largely agreed with conventional extraction-based diagnostics targeting SARS-CoV-2 genes E and RdRP ( Fig. 3c and Table 2 ). That C T values from hid-RT-qPCR overall were inferior (higher) compared to conventional extraction-based RT-qPCR detection however suggest a lower analytical sensitivity of the hid-RT-qPCR method. To further improve the sensitivity of SARS-CoV-2 hid-RT-qPCR, the heatinactivation procedure should promptly be optimized to maximize the RNA integrity of the sample while effectively inactivating the virus. We and others [10] found that brief inactivation at high temperature (95°C) provided higher SARS-CoV-2 RNA yield compared to longer heat-inactivation at lower temperature. In addition, we suggest that by adding the chelator EDTA before heating, metal-ion-based RNA cleavage during heating could be reduced. Supplement of MgCl 2 in the RT-qPCR reaction may then compensate for lowered available Mg 2+ , needed for reverse transcription. Moreover, the medium could be buffered to lowered pH to better preserve the phosphodiester bond of RNA during heating, and, chemical (heat resistant) RNAse inhibitors may further increase sensitivity in hid-RT-qPCR. In addition to being heat resistant, readily available chemical RNase inhibitors are cheap, making them suitable for scaled diagnostics (although their effect on RT-qPCR need to be characterized). We and others should combine our efforts to identify the optimal heat-inactivation formula for SARS-CoV-2 hid-RT-qPCR.