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

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

doi:10.1021/acsomega.0c05635

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.

Highlights

In December 2019, an outbreak of a new syndrome characterized by serious symptoms including fever, severe respiratory illness, and acute pneumonia, eventually leading to respiratory failure and death, was reported in the Wuhan city of Hubei Province in China
Because our His-Thermus aquaticus DNA polymerase (Taq Pol) has the linker peptide at the N-terminus fused to the histidine tag, it migrated at a slower rate than native Taq Pol
We evaluated the performance of our R3T one-step qRT-polymerase chain reaction (PCR) kit in a Saudi Ministry of Health-approved testing facility using 192 patient samples who were screened for SARS-CoV-2 infection

Summary

Introduction

In December 2019, an outbreak of a new syndrome characterized by serious symptoms including fever, severe respiratory illness, and acute pneumonia, eventually leading to respiratory failure and death, was reported in the Wuhan city of Hubei Province in China. The COVID-19 pandemic is caused by the new strain (SARS-CoV-2) classified under the genus betacoronavirus and the subgenus sarbecovirus.[5,6] A large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in bats, which are their natural hosts.[7−9] SARS-CoV-2 is 96% identical at the whole genome level to bat CoV and shares 79.6% sequence identity to SARS-CoV.[5,10] Coronaviruses are characterized by large, single-stranded, positive-sense RNA genomes ranging from 26 to 32 kb.[11] Coronaviruses express their replication and transcription complexes, including RNAdependent RNA polymerase (RdRp), from a single large open reading frame referred to as ORF1ab.[12] The viral particle is composed of four main structural proteins, spike (S), Received: November 19, 2020 Accepted: March 1, 2021 Published: March 15, 2021

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