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

Using synthetic RNA template of the SARS-CoV-2 N gene target, the LoD of standard RT-qPCR was assessed to be 120 RNA copies per reaction with a Cq mean value of 40.67 ± 0.29. However, no amplification was observed when RNA was spiked in sputum and nasal exudate using this standard mastermix. Using a PCR inhibitor-resistant mastermix, the LoD of DIRECT-PCR was determined to be 120 RNA copies per reaction with a Cq mean value of 38.48 ± 0.57 at the 7 th order of magnitude ( Table 1 ). The lowest Cq was observed at 17.37 ± 0.04 which corresponded to 1.2 x 10 8 RNA copies per reaction (Fig. 1A) . The DIRECT-PCR assay was completed in 72 minutes on the benchtop thermocycler. The control assay has amplification efficiency (E) of 84.94% with a correlation coefficient (R 2 ) of 0.9884 (Fig. 1D) . No amplification was observed for the NTC.