TY - JOUR
T1 - Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes
AU - Takahashi, Masateru
AU - Tehseen, Muhammad
AU - Salunke, Rahul Pandurang
AU - Takahashi, Etsuko
AU - Mfarrej, Sara
AU - Sobhy, Mohamed Abdelmaboud
AU - Alhamlan, Fatimah S.
AU - Hala, Sharif
AU - Ramos Mandujano, Gerardo
AU - Al-Qahtani, Ahmed A.
AU - Alofi, Fadwa S.
AU - Alsomali, Afrah
AU - Hashem, Anwar M.
AU - Khogeer, Asim
AU - Almontashiri, Naif A. M.
AU - Lee, Jae Man
AU - Mon, Hiroaki
AU - Sakashita, Kosuke
AU - Li, Mo
AU - Kusakabe, Takahiro
AU - Pain, Arnab
AU - Hamdan, Samir
N1 - KAUST Repository Item: Exported on 2021-03-25
Acknowledged KAUST grant number(s): BAS/1/1020-01-01
Acknowledgements: 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.
PY - 2021/3/15
Y1 - 2021/3/15
N2 - 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.
AB - 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.
UR - http://hdl.handle.net/10754/668245
UR - https://pubs.acs.org/doi/10.1021/acsomega.0c05635
U2 - 10.1021/acsomega.0c05635
DO - 10.1021/acsomega.0c05635
M3 - Article
C2 - 33778250
SN - 2470-1343
JO - ACS Omega
JF - ACS Omega
ER -