Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes
Salunke, Rahul Pandurang
Sobhy, Mohamed Abdelmaboud
Alhamlan, Fatimah S.
Ramos Mandujano, Gerardo
Al-Qahtani, Ahmed A.
Alofi, Fadwa S.
Hashem, Anwar M.
Almontashiri, Naif A. M.
Lee, Jae Man
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
Laboratory of DNA Replication and Recombination
Pathogen Genomics Laboratory
Proteomics, protein expression & cytomet
KAUST Grant NumberBAS/1/1020-01-01
Permanent link to this recordhttp://hdl.handle.net/10754/668245
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AbstractOne-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.
CitationTakahashi, M., Tehseen, M., Salunke, R., Takahashi, E., Mfarrej, S., Sobhy, M. A., … Hamdan, S. M. (2021). Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes. ACS Omega. doi:10.1021/acsomega.0c05635
SponsorsThis research was funded by baseline funding from KAUST to S.M.H. and COVID-19 response initiative by the Vice President of research at KAUST. The clinical COVID-19 samples were collected as part of KAUST baseline funding (BAS/1/1020-01-01) to AP and the R3T initiative by the the Vice President of research at KAUST. The authors declare no conflicts of interest.
PublisherAmerican Chemical Society (ACS)
Except where otherwise noted, this item's license is described as This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Omega, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://pubs.acs.org/doi/10.1021/acsomega.0c05635.