An Ultrafast One-Step Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Detection of SARS-CoV-2.

Jadranka Milosevic,Mengrou Lu,Si-Yang Zheng,Hong-Zhang He,Wallace Greene

doi:10.3389/fmicb.2021.749783

Jadranka Milosevic, Mengrou Lu + Show 3 more

Open Access

https://doi.org/10.3389/fmicb.2021.749783

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Abstract

We developed an ultrafast one-step RT-qPCR assay for SARS-CoV-2 detection, which can be completed in only 30 min on benchtop Bio-Rad CFX96. The assay significantly reduces the running time of conventional RT-qPCR: reduced RT step from 10 to 1 min, and reduced the PCR cycle of denaturation from 10 to 1 s and extension from 30 to 1 s. A cohort of 60 nasopharyngeal swab samples testing showed that the assay had a clinical sensitivity of 100% and a clinical specificity of 100%.

Highlights

The current highly transmissible outbreak of severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the leading cause of morbidity and mortality across the globe (Andrasfay and Goldman, 2021; Cohen, 2021; Woolf et al, 2021)
We described an ultrafast one-step quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assay for the qualitative detection of SARS-CoV-2 that is fully compatible with conventional benchtop qPCR instruments
In developing the ultrafast one-step qRT-PCR assay, we reasoned that the enzymes in the qRT-PCR are key to significantly shortening the qRT-PCR and to keeping comparable sensitivity as conventional qRT-PCR for SARS-CoV-2 detection

Summary

Introduction

The current highly transmissible outbreak of severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the leading cause of morbidity and mortality across the globe (Andrasfay and Goldman, 2021; Cohen, 2021; Woolf et al, 2021). Researchers have intensively invested in developing innovation for cost-effective point-of-care test kits and efficient laboratory techniques for confirmation of SARS-CoV-2 infection (Carter et al, 2020; Chen et al, 2020; Shuren and Stenzel, 2020; Venter and Richter, 2020; Wiersinga et al, 2020; El Jaddaoui et al, 2021; Mardian et al, 2021; Taleghani and Taghipour, 2021; Vandenberg et al, 2021; Yüce et al, 2021) Among those technologies, real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) of nasopharyngeal swabs is the current gold standard in the clinical setting to confirm the clinical diagnosis of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Carter et al, 2020; Ji et al, 2020; Tang et al, 2020; Kevadiya et al, 2021). To meet the pandemic challenges, it is important to significantly shorten the turnaround time in the race for increasing the number of diagnostic tests

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