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

An even more direct route for SARS-CoV-2 testing might be to run RT-qPCR on unpurified swabs samples, or even on saliva, lysed directly before RT-qPCR. SARS coronavirus capsids are self-assembled particles in which the lipid bilayer is a weak spot [12], thus the viral capsid can be ruptured by surfactants and at the same time viral RNA can be released from similarly lysed human cells in the sample [13] . RT-qPCR assays directly on detergent-inactivated samples would require an RT-qPCR assay resilient to high concentrations of detergent. We determined the effect of Triton-X100 and Tween-20, on SARS-CoV-2 RT-qPCR, using a spike-in of 50,000 copies synthetic full-genome SARS-CoV-2 RNA (SKU102024-MN908947.3, Twist Biosciences) and the N1 primerprobe set. We found that C T values were only modestly affected (+1-2 CT) when incubated with as much as 5% Triton X-100 or 10% Tween-20 in the RT-qPCR reaction ( Fig. 4a-b ). We observed a lowered level of fluorescence in the plateau phase in qPCR at increased concentrations of detergent, without markedly affecting the CT (Fig. 4c-d) . As a step to test whether SARS-CoV-2 could be detected after direct lysis, we obtained six aliquots of saliva and six combined nose and throat swabs in PBS from six deidentified donors (Methods), of which four had been identified as COVID-19 positive and two as negative in extractionbased routine diagnostics (sample aliquots obtained from the Public Health Agency of Sweden, Folkhälsomyndigheten). Notably, these samples were not collected by health care professionals using clinical grade flocked plastic swabs, rather the samples were self-collected using simple cotton swabs (deposition in PBS) and a jar without storage buffer for saliva (Methods). Furthermore, at the time of our experiment, these samples had been freeze-thawed and stored at room temperature for several hours combined. We tested these samples blindly, by mixing 5μl sample (saliva or nose + throat swab) with 5μl of 10 or 20% Triton X-100 and performing the SARS-CoV-2 RT-qPCR using the N1 primers and probe directly on these lysates. Indeed, all four COVID-19 positive donor individuals were correctly called as SARS-CoV-2 positive in at least one Triton X-100 condition or sample (saliva and/or nose + throat swab), while negative controls lacked signal (Fig. 4e) .