Open-source real-time quantitative RT-PCR-based on a RNA standard for the assessment of SARS-CoV-2 viral load

Abstract

BACKGROUND Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes by molecular methods has been chosen as the main approach to identify individuals with Coronavirus disease 2019 (COVID-19) infection.OBJECTIVES In this study, we developed an open-source RNA standard-based real-time quantitative RT-PCR (RT-qPCR) assay for quantitative diagnostics of SARS-CoV-2 from nasopharynx, oropharynx, saliva and plasma samples. METHODS AND FINDINGSWe evaluated three SARS-CoV-2 target genes and selected the RNA-dependent RNA polymerase (RdRp) gene, given its better performance. To improve the efficiency of the assay, a primer gradient containing 25 primers forward and reverse concentration combinations was performed. The forward and reverse primer pairs with 400 nM and 500 nM concentrations, respectively, showed the highest sensitivity. The LOD 95% was ~60 copies per reaction. From the four biological matrices tested, none of them interfered with the viral load measurement. Comparison with the Allplex TM 2019-nCoV assay (Seegene) demonstrated that our test presents 90% sensitivity and 100% specificity. MAIN CONCLUSIONSWe developed an efficient molecular method able to measure absolute SARS-CoV-2 viral load with high replicability, sensitivity and specificity in different clinical samples. Key words: reverse transcriptase polymerase chain reaction -real-time polymerase chain reaction -SARS-CoV-2 -COVID-19 -viral load -COVID-19 testingDiagnostic molecular assays have been playing an essential role in the pandemic scenario of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), monitoring the suspected and confirmed Coronavirus disease 2019 (COVID-19) cases and the dynamics of virus infection. Given this, these analyses are invaluable resources to guide decision-making of the public healthcare system. The gold standard for SARS-CoV-2 diagnosis is real-time reverse transcription polymerase chain reaction (RT-PCR), which is a sensitive method that can rapidly confirm an infection a few days after exposure. (1) However, the method is often qualitative (i.e., only providing informing about the presence/absence of the virus but not its quantity). The evaluation of the viral load could aid the understanding of the course of the pathogenic process and therefore could provide information for the management of the infection. (2)