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

Masateru Takahashi, Muhammad Tehseen, Rahul Pandurang Salunke, Etsuko Takahashi, Sara Mfarrej, Mohamed Abdelmaboud Sobhy, Fatimah S. Alhamlan, Sharif Hala, Gerardo Ramos Mandujano, Ahmed A. Al-Qahtani, Fadwa S. Alofi, Afrah Alsomali, Anwar M. Hashem, Asim Khogeer, Naif A. M. Almontashiri, Jae Man Lee, Hiroaki Mon, Kosuke Sakashita, Mo Li, Takahiro KusakabeArnab Pain, Samir Hamdan

Research output: Contribution to journalArticlepeer-review

Abstract

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.
Original languageEnglish (US)
JournalACS Omega
DOIs
StatePublished - Mar 15 2021

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