A new SYBR Green real-time PCR to detect SARS-CoV-2

D R Marinowic,A M Alcará,F V F Rodrigues,G Zanirati,D C Machado,J C Da Costa,M V C Grahl

doi:10.1038/s41598-021-81245-0

Abstract

Phylogenetic analysis has demonstrated that the etiologic agent of the 2020 pandemic outbreak is a betacoronavirus named SARS-CoV-2. For public health interventions, a diagnostic test with high sensitivity and specificity is required. The gold standard protocol for diagnosis by the Word Health Organization (WHO) is RT-PCR. To detect low viral loads and perform large-scale screening, a low-cost diagnostic test is necessary. Here, we developed a cost-effective test capable of detecting SARS-CoV-2. We validated an auxiliary protocol for molecular diagnosis with the SYBR Green RT-PCR methodology to successfully screen negative cases of SARS-CoV-2. Our results revealed a set of primers with high specificity and no homology with other viruses from the Coronovideae family or human respiratory tract pathogenic viruses, presenting with complementarity only for rhinoviruses/enteroviruses and Legionella spp. Optimization of the annealing temperature and polymerization time led to a high specificity in the PCR products. We have developed a more affordable and swift methodology for negative SARS-CoV-2 screening. This methodology can be applied on a large scale to soften panic and economic burden through guidance for isolation strategies.

Highlights

In December 2019, an outbreak of pneumonia with unknown etiology was identified in Wuhan, China
According to the World Health Organization (WHO), the gold standard method to detect Severe Acute Respiratory Syndrome (SARS)-CoV-2 is real-time polymerase chain reaction (RT-PCR) using TaqMan probes, which precisely detect the presence of the v irus[10]
We developed an auxiliary protocol for molecular diagnosis involving RT-PCR with a SYBR Green methodology to detect negative cases of SARS-CoV-2

Summary

Introduction

In December 2019, an outbreak of pneumonia with unknown etiology was identified in Wuhan, China. Phylogenetic analyses have proven that the etiologic agent of the Wuhan pneumonia outbreak is a betacoronavirus called SARS-CoV-24. Low-cost diagnostic tests must be developed for large-scale patient screening to confirm positive and/or negative cases of the new coronavirus. To this end, we developed an auxiliary protocol for molecular diagnosis involving RT-PCR with a SYBR Green methodology to detect negative cases of SARS-CoV-2. We developed an auxiliary protocol for molecular diagnosis involving RT-PCR with a SYBR Green methodology to detect negative cases of SARS-CoV-2 This protocol will maximize the cost–benefit of viral detection and accelerate availability by the use of conventional kits on a large scale in molecular biology laboratories

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Conclusion