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

A commercially available portable qPCR thermocycler was validated by comparing to the benchtop thermocycler using the fast DIRECT-PCR assay of RNA and plasmid templates spiked in sputum and nasal exudate. The amplification efficiency of RNA control showed that there was no significant difference (p-value = 0.4344) between benchtop and portable thermocycler. Both thermocyclers had similar LoD at 600 RNA copies per reaction. When using RNA spike-in sputum, the portable thermocycler had LoD of 600 RNA copies per reaction (Cq of 36.25 ± 0.46) compared to 6 RNA copies per reaction when using the benchtop thermocycler (Table 1) . On the other hand, both thermocyclers had the same LoD of 60 RNA copies per reaction when using RNA spike-in nasal exudate (Table 1) . Using plasmid controls, there was no significant difference (p-value = 0.7799) in amplification efficiency between both thermocyclers (Fig. 2) . Next, using plasmids encoding the N gene of SARS-CoV-2 as control, the LoD was found to be 2 and 20 copies per reaction when using plasmid template and plasmid spike-in sputum for benchtop and portable thermocycler respectively. Both thermocyclers had similar sensitivity of 20 copies per reaction when using plasmid spike-in nasal exudate (Table 1 ). In terms of duration, the reaction took 49 minutes on the portable thermocycler and 36 minutes on benchtop thermocycler.