Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler

Soon Keong Wee; Suppiah Paramalingam Sivalingam; Eric Peng Huat Yap

To reduce the duration and cost of the DIRECT-PCR assay, we modified the total reaction volume to 10 µL and duration of cycling conditions with the addition of 1 µL template. Amplification of RNA template using the fast DIRECT-PCR demonstrated detection at 600 RNA copies per reaction (Cq of 39.42) in contrast to 120 RNA copies per reaction (Cq of 38.48 ± 0.57) detected on the DIRECT-PCR assay. This fast DIRECT-PCR was completed in 36 minutes as compared to 72 minutes for the DIRECT-PCR assay. The LoD was similar at 6 (Cq of 39.28) and 60 RNA copies per reaction (Cq of 39.34) when spiked into sputum and nasal exudate samples respectively (Table 1 ). Using positive control plasmids encoding the N gene of SARS-CoV-2, the LoD was found to be 2 copies per reaction (Cq of 39.75) using the benchtop thermocycler. LoD of 2 and 20 copies per reaction was observed when the plasmids were spiked into sputum and nasal exudate samples with Cq of 38.93 and Cq mean of 38.02 ± 0.55 respectively over four orders of magnitude (Fig. 2) . No amplification was observed for the negative controls using MERS-CoV and SARS-CoV plasmids. Human RP primers were used as an internal control to amplify and detect the presence of human RNA present in the crude samples (data not shown). Using crude sputum and nasal exudate directly, the mean Cq values for human RP was 26.12 ± 0.24 and 27.76 ± 0.81 respectively on the benchtop thermocycler. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.17.042366 doi: bioRxiv preprint