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

• Published in: ACS Omega Vol. 6; no. 11; pp. 7374 - 7386
• Main Authors: Takahashi, Masateru, Tehseen, Muhammad, Salunke, Rahul, Takahashi, Etsuko, Mfarrej, Sara, Sobhy, Mohamed A, Alhamlan, Fatimah S, Hala, Sharif, Ramos-Mandujano, Gerardo, Al-Qahtani, Ahmed A, Alofi, Fadwa S, Alsomali, Afrah, Hashem, Anwar M, Khogeer, Asim, Almontashiri, Naif A. M, Lee, Jae Man, Mon, Hiroaki, Sakashita, Kosuke, Li, Mo, Kusakabe, Takahiro, Pain, Arnab, Hamdan, Samir M
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 23.03.2021; American Chemical Society (ACS)
• Subjects: 3T; Biochemistry; Biophysics; Biotechnology; Chemistry; Computational Biology; COVID -19; COVID -19 Diagnostics; Developmental Biology; enzyme production strategies; Genetics; Health-approved testing facility; Immunology; In-House Enzymes One-step reverse-t; Infectious Diseases; Invitrogen SuperScript III Platinum; Molecular Biology; Moloney murine leukemia virus; One-Step qRT-PCR Kit; polymerase chain reaction; QD1-999; Rapid Research Response Team; RNA; Thermus aquaticus DNA polymerase; Virology
• ISSN: 2470-1343; 2470-1343
• DOI: 10.1021/acsomega.0c05635

One-step reverse-transcription quantitative polymerase chain reaction (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 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 SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility.