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

Quantitative RT-PCR is being deployed as primary method for SARS-CoV-2 detection in research and hospital laboratories on account of its singlemolecule sensitivity, ease of assay design and availability of reagents. However, there are several technical challenges. These tests require RNA extraction, followed by amplification and detection. Current state-of-art PCR typically require 70 minutes for RNA extraction and a further 90 minutes for amplification (15, 16) . Highly trained technical staff, costly equipment (costing USD 20K -50K) and facilities are required. These factors contribute to longer turnaround-time, costs of manpower, capital and consumables as well as risks of carryover contamination and biosafety risks when handling clinical samples. There is a limited supply of extraction reagents and test kits worldwide (16, 17) . In addition, asymptomatic individuals have also led to pre-symptomatic transmission in the community which further increases demand for laboratory testing (13) . These factors have motivated us to explore ways to simplify and shorten the protocols, without significant compromise to the high sensitivity and specificity of RT-qPCR. We therefore propose a novel method, which we termed direct rapid extraction-free PCR (DIRECT-PCR) for Covid-19 diagnosis.