The Potential Use of Unprocessed Sample for RT-qPCR Detection of COVID-19 without an RNA Extraction Step

Arunkumar Arumugam; Season S Wong

We next tested whether the RNA from SARS-CoV-2 can be detected by directly spiking samples of the non-replicative recombinant virus particles (SeraCare AccuPlex SARS-CoV-2 reference material) in VTM to master mix without an extraction step. The SARS-CoV-2 virus particles were mixed with VTM to get a final concentration of 2,500 copies per mL. Different amounts (2, 4, 6 and 8 µL) of these mock clinical samples were spiked into the master mix containing CDC recommended SARS-CoV-2 RT-qPCR diagnostic panel primers N1, N2 o N3 in 20 µL PCR reactions, though we note that the N3 primers were recently removed by the CDC. Purified nucleic acid template (4 µL) isolated from a Promega Maxwell device was also amplified. The Ct difference between the purified template and the directly spiked clinical samples (i.e., 4 µL, 5 µL and 6 µL) is minimal.