R3T (Rapid Research Response Team) One-step RT-qPCR kit for COVID-19 diagnostic using in-house enzymes

Takahashi, M.; Tehseen, M.; Salunke, R.; Takahashi, E.; Mfarrej, S.; Sobhy, M. A.; Alhamlan, F.; Hala, S.; Mandujano, G. R.; Al-Qahtani, A. A.; Alofi, F. S.; Alsomali, A.; Hashem, A. M.; Khogeer, A.; Almontashiri, N. A. M.; Lee, J. M.; Mon, H.; Sakashita, K.; Li, M.; Kusakabe, T.; Pain, A.; Hamdan, S. M.

One-step RT-qPCR is the most widely applied method for COVID-19 diagnostics. Designing in-house one-step RT-qPCR kits is restricted by the patent-rights for the production of enzymes and the lack of information about the components of the commercial kits. Here, we provide a simple, economical, and powerful one-step RT-qPCR kit based on patent-free, specifically-tailored versions of Moloney Murine Leukemia Virus Reverse Transcriptase and Thermus aquaticus DNA polymerase termed the R3T (Rapid Research Response Team) One-step RT-qPCR. 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 RT-qPCR and TaqPath 1-Step RT-qPCR kits. Overall, our kit has shown robust performance in both of laboratory settings and the Saudi Ministry of Health-approved testing facility.