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

Next, the DIRECT-PCR assay was evaluated using RNA spiked in sputum and nasal exudate matrices. The amplification of spiked RNA with PCR inhibition tolerance was observed with the concentration of sputum and nasal exudate at 4.25% (v/v) in the PCR reaction. In RNA spiked sputum samples, the sensitivity was greater than the control at 12 RNA copies per reaction with a Cq of 38.79 (Table 1 ). The lowest Cq was observed at 16.22 ± 0.24 which corresponded to 1.2 x 10 8 RNA copies per reaction (Fig. 1B) . When RNA was spiked in nasal exudate, we found the LoD of 12 RNA copies per reaction with a Cq of 38.72 (Table 1) . These LoDs were achieved in one out of two duplicates tested. The lowest Cq in nasal exudate was observed at 15.18 ± 0.36 which corresponded to 1.2 x 10 8 RNA copies per reaction (Fig. 1C) . The PCR efficiency revealed lower amplification efficiency (E) when spiked into sputum (82.62%) and nasal exudate (81.27%) compared to control while the correlation coefficient (R 2 ) at 0.9944 and 0.9986 for sputum and nasal exudate respectively. The linear regression lines had similar slopes (p-value = 0.6503) while the intercepts were significantly different (p-value < 0.0001) (Fig. 1D) .