Quick and Easy Assembly of a One-step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes

Abstract

One-step qRT-PCR is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well-suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT- PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically-tailored versions of Moloney Murine Leukemia Virus Reverse Transcriptase and Thermus aquaticus DNA polymerase and termed the R3T (Rapid Research Response Team) One-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of the SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity as that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath 1-Step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility.