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

Our dilution experiments of medium and spike-in synthetic SARS-CoV-2 RNA had shown limited inhibition at ≤25% medium in the reaction (Fig. 2d) . However, clinical samples contain additional material from the swab and other unknown and potentially inhibitory agents. In addition, due to potentially large variability between clinical samples, it is important to characterize inhibition curves in multiple individual clinical samples rather than an averaged mix of samples. The optimal amount of sample input in hid-RT-qPCR should be a balance between possible inhibition from the sample and the amount of RNA going into the reaction. To identify the optimal range of sample input in clinical samples, we performed dilution series (10 to 0.01μl) of individual COVID-19 positive heat-inactivated (65°C, 30 min) nasopharyngeal swab samples, and found an input of 4-1μl sample in a 20μl TaqPath RT-qPCR to be optimal, avoiding the sharp inhibitory effect at higher amounts of sample input observed in some individual samples, yet minimizing C T (Fig. 2i-j) .