Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes
Type
ArticleAuthors
Takahashi, MasateruTehseen, Muhammad

Salunke, Rahul Pandurang
Takahashi, Etsuko
Mfarrej, Sara
Sobhy, Mohamed Abdelmaboud
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

KAUST Department
Biological and Environmental Sciences and Engineering (BESE) DivisionBioscience Program
Laboratory of DNA Replication and Recombination
Pathogen Genomics Laboratory
Proteomics, protein expression & cytomet
KAUST Grant Number
BAS/1/1020-01-01Date
2021-03-15Submitted Date
2020-11-19Permanent link to this record
http://hdl.handle.net/10754/668245
Metadata
Show full item recordAbstract
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.Citation
Takahashi, 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.0c05635Sponsors
This 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.Publisher
American Chemical Society (ACS)Journal
ACS OmegaAdditional Links
https://pubs.acs.org/doi/10.1021/acsomega.0c05635ae974a485f413a2113503eed53cd6c53
10.1021/acsomega.0c05635
Scopus Count
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.