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Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes.
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.
  • Takahashi M; Laboratory of DNA Replication and Recombination, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
  • Tehseen M; Laboratory of DNA Replication and Recombination, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
  • Salunke R; Pathogen Genomics Laboratory, BESE Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
  • Takahashi E; Laboratory of DNA Replication and Recombination, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
  • Mfarrej S; Pathogen Genomics Laboratory, BESE Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
  • Sobhy MA; Laboratory of DNA Replication and Recombination, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
  • Alhamlan FS; Department of Infection and Immunity, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia.
  • Hala S; Pathogen Genomics Laboratory, BESE Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
  • Ramos-Mandujano G; King Saud Bin Abdulaziz University of Health Sciences, Jeddah 22384, Saudi Arabia.
  • Al-Qahtani AA; King Abdullah International Medical Research Centre, Jeddah, Makkah, Ministry of National Guard Health Affairs, Jeddah, Makkah 22384, Saudi Arabia.
  • Alofi FS; Stem Cell and Regenration Laboratory. Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
  • Alsomali A; Department of Infection and Immunity, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia.
  • Hashem AM; Infectious Diseases Department, King Fahad Hospital, Madinah 3177, Saudi Arabia.
  • Khogeer A; King Abdullah Medical Complex (KAMC), Jeddah 23816, Saudi Arabia.
  • Almontashiri NAM; Vaccines and Immunotherapy Unit, King Fahd Medical Research Center; King Abdulaziz University, Jeddah, Saudi Arabia.
  • Lee JM; Department of Medical Microbiology and Parasitology, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
  • Mon H; Plan and Research Department, General Directorate of Health Affairs Makkah Region, MOH Mecca 24321, Saudi Arabia.
  • Sakashita K; College of Applied Medical Sciences, Taibah University, Madinah 41311, Saudi Arabia.
  • Li M; Center for Genetics and Inherited Diseases, Taibah University, Madinah 42353, Saudi Arabia.
  • Kusakabe T; Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan.
  • Pain A; Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan.
  • Hamdan SM; Department of Infection and Immunity, King Faisal Specialist Hospital and Research Centre, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
ACS Omega ; 6(11): 7374-7386, 2021 Mar 23.
Article in English | MEDLINE | ID: covidwho-1155691
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.

Full text: Available Collection: International databases Database: MEDLINE Type of study: Prognostic study Language: English Journal: ACS Omega Year: 2021 Document Type: Article Affiliation country: Acsomega.0c05635

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Full text: Available Collection: International databases Database: MEDLINE Type of study: Prognostic study Language: English Journal: ACS Omega Year: 2021 Document Type: Article Affiliation country: Acsomega.0c05635