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

Current RT-qPCR for SARS-CoV-2 involves RNA purification as part of the pre-PCR sample The copyright holder for this preprint (which was not peer-reviewed) is the .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 preparation procedure and at least four to six hours are needed for time-to-results in most laboratories (16) . When the cycling conditions were optimized in our fast DIRECT-PCR with small reaction volumes (10 µL), and shorter RT and annealing durations, we were able to amplify SARS-CoV-2 in less than an hour (Fig. 3) . Furthermore, there are cost savings on nucleic acid extraction kits, and the availability of reagents themselves may be limiting when laboratory capacity is ramped up and demand is increased globally. Moreover, DIRECT-PCR is a single-tube homogeneous reaction that reduces hands-on time and biosafety risk for laboratory personnel, as well as the likelihood for carryover contamination. One caveat however for fast DIRECT-PCR of samples with low viral load is that of sampling error, since only 1 µL of sample is used. Nucleic acid extractions, on the other hand, serve to concentrate RNA from typical sample volumes of 150-300 µL, although their yield can also be low and variable. Hence, where samples are expected to have low counts near to the limit of detection, DIRECT-PCR with larger volume reactions (25 µL) to include higher template volume may be necessary to reduce risk of false negatives.