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 FINDINGS We 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 CONCLUSIONS We developed an efficient molecular method able to measure absolute SARS-CoV-2 viral load with high replicability, sensitivity and specificity in different clinical samples.
【저자키워드】 COVID-19, SARS-CoV-2, COVID-19 testing, Reverse transcriptase polymerase chain reaction, Viral load, real-time polymerase chain reaction, 【초록키워드】 coronavirus disease, Saliva, Coronavirus disease 2019, coronavirus, Infection, detection, severe acute respiratory syndrome Coronavirus, RT-PCR, diagnostics, RNA, clinical samples, sensitivity, specificity, RT-qPCR, Viral, Sensitivity and specificity, Viral load, nasopharynx, RdRP, RNA-dependent RNA polymerase, molecular, respiratory, Quantitative, Target genes, Combination, Concentration, Efficiency, target gene, (RdRp, acute respiratory syndrome, reverse primer, acute respiratory syndrome coronavirus, acute respiratory syndrome coronavirus 2, reaction, individual, plasma samples, SARS-CoV-2 viral load, primer, 2019-nCoV assay, oropharynx, approach, concentrations, IMPROVE, selected, highest, tested, identify, evaluated, was performed, demonstrated, primer pair, 【제목키워드】 RNA, Quantitative, SARS-CoV-2 viral load,