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 further evaluate whether direct RT-qPCR on heatinactivated samples might allow effective and sensitive COVID-19 diagnostics, we performed heat inactivation (65°C, 30 min) of aliquots from 85 clinically diagnosed nasopharyngeal samples and performed hid-RT-qPCR blindly to their COVID-19 status. For all samples, we used 4μl input and primers N1, RdRP, as well as RNase P for assessment of sample RNA integrity. Thereafter, we combined the results of hid-RT-qPCR with CT values from the clinical diagnostics performed on extracted RNA (MagNA Pure 96, Roche Diagnostics, test targeting E and RdRP) (Fig. 3) . We observed strong correlation between CT values of extracted and heat-inactivated samples (Fig. 3a) . CT values for hid-RT-qPCR samples were higher than for RNA eluates of the same samples (median 6.7 Ct difference) (Fig. 3b) . This was expected given that (1) More RNA was loaded for eluates (2.5x, standard 10μl input vs. 4μl volume equivalent in hid-RT-qPCR), (2) RNA extraction of eluates was performed on fresh samples while the aliquots used for hid-RT-qPCR had been frozen and stored at -20°C, and then thawed before heat inactivation, (3) Heating may degrade RNA in presence of RNases and/or metal ions (metal-ionbased RNA cleavage), and (4) Inhibitory agents from the swab and medium may inhibit RT-qPCR, although significant medium inhibition is unlikely (Fig. 2d,i-j) . By performing RNA re-extraction from 19 freeze-thawed aliquots and comparing C T values to eluates of matched fresh samples we found the effect of freezethaw to result in +2-3 CT (Fig. 3b) . However, difference in CT value is not the same as difference in diagnostic outcome (i.e. detected presence or absence of SARS-CoV-2). We plotted a heatmap of CT values (Fig. 3c) and found a striking agreement between clinical diagnostics performed on RNA eluates and hid-RT-qPCR COVID-19 calls. Absent-or-present calls in qPCR analysis is dependent on the arbitrary cycle-number cut-off applied, and high cut-off may be valid especially if the PCR efficiency is expected to be low in individual samples. On the other hand, only one of the positive samples in hid-RT-qPCR had a detected SARS-CoV-2 amplification recorded with CT above 40 (Fig. 3c , sample 63). Notably, using diagnostic of extracted RNA and best-performing target RdRP as reference, hid-RT-qPCR using primer-probe set N1 showed an accuracy of 92% (95% confidence interval [CI95%]: 84-97%, P= 1.6 x 10 -15 , binominal test; 91% sensitivity; 93% specificity) ( Table 2) . Among the 85 cases, hid-RT-qPCR using N1 + RdRP missed three COVID-19 positive samples that were detected by routine diagnostics on RNA eluates targeting genes E + RdRP. Interestingly, three samples were identified as COVID-19 positive by All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.