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

The DIRECT-PCR assay was modified in order to further reduce turnaround time and reagent cost. The final reaction volume was reduced from 20 µL to 10 µL, while reducing the number of cycles from 45 to 40, reducing reverse transcription (RT) step from 15 minutes to 5 minutes, reducing initial denaturation from 1 minute to 30 seconds and reducing annealing duration from 45 seconds to 15 seconds. Ten-fold serially diluted RNA spike-in matrices described above were used in duplicates to evaluate the modified assay to determine a fast protocol for detection of SARS-CoV-2. Briefly, an aliquot of 1 μL of template was added in the 10 μL PCR reaction mix mentioned above. Similarly, the plasmid controls of N gene from SARS-CoV-2, MERS-CoV and SARS-CoV were ten-fold serially diluted in DNase/RNasefree water to achieve 6 orders of magnitude respectively. Subsequently, 1 µL of diluted plasmid template was spiked into the sputum and nasal exudate mix containing Sputasol and RNaseOUT forming a total volume of 10 µL as described above. The internal control using human Ribonuclease P (RP) gene was evaluated by the DIRECT-PCR of human RP in the sputum and nasal exudate using the . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 assay described. Serially diluted human RP RNA template in water was used as the positive control. MERS-Cov and SARS-Cov plasmid controls were added as negative controls. The amplification was performed on a standard benchtop real-time thermocycler (StepOnePlus Real-Time PCR System, Applied Biosystems, USA).