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

Arunkumar Arumugam; Season S Wong

We also tested VTM mixed SARS-CoV-2 plasmid (purchased from Integrated DNA Technologies, Inc.) using RT-qPCR. This plasmid is used as a positive control for the CDC's SARS-CoV-2 RT-qPCR assay. The positive control plasmid was mixed with VTM and 4 µL of this mix was used for the RT-qPCR reaction. The RT-qPCR results showed that the Ct values of control (without VTM) and VTM mixed reactions (all containing 400 copies/reaction) were very similar for all three (N1, N2 and N3) SARS-CoV-2 targets ( Table 3) . We also tested 40 copies of SARS-CoV-2 plasmid using RT-qPCR, which gave a Ct value of ~38 (data not shown). This means direct spiking of a higher concentration of SARS-CoV-2 genome copies in VTM did not have a major PCR inhibitory effect in the reactions when the target concentration is high ( Table 3) . Therefore, it is likely that specimens with higher viral load could also be detected by RT-qPCR without needing an RNA extraction step. SARS-CoV-2 plasmid in VTM did not show any PCR inhibition when compared to plasmid alone. The CDC positive control plasmid for SARS-CoV-2 (from IDT) was diluted in VTM or TE buffer (control) to get 100,000 copies per mL. 4 µL of SARS-CoV-2 plasmids in VTM and in TE buffer were added to a 20 µL PCR reaction mix targeting N1, N2 and N3 genes for the detection of SARS-CoV-2. The Ct values of both the control and plasmid in VTM are very similar, indicating that the sample preparation step can be omitted in high viral load samples.