Validation of Real-Time RT-PCR for Detection of SARS-CoV-2 in the Early Stages of the COVID-19 Outbreak Without Emergency Use Authorization Reagents in the Republic of Korea

Abstract Background: A real-time reverse transcription polymerase chain reaction (RT-PCR) assay for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was developed to rapidly diagnose coronavirus disease 2019 (COVID-19). Early diagnosis of COVID-19 enables timely treatment and the implementation of public health measures. We validated the sensitivity, specificity, precision, linearity, accuracy, and robustness of a real-time RT-PCR-based assay for SARS-CoV-2 detection and compared the sensitivity and specificity of Emergency Use Authorization (EUA)-approved reagents for COVID-19 with those of the real-time RT-PCR method.Methods: The real-time RT-PCR-based assay was performed to specifically amplify genomic markers of SARS-CoV-2.Results: The real-time RT-PCR assay was highly specific for SARS-CoV-2, and did not amplify genomic fragments of 13 other viruses that cause respiratory diseases. The assay showed high linearity when conducted with a viral isolate from a patient with COVID-19 together with plasmids containing the target genes. The assay showed good repeatability and reproducibility, with a coefficient of variation of 3%, and detected SARS-CoV-2 with a limit of detection of 1 PFU/mL.Conclusions: The present real-time RT-PCR-based assay can diagnose COVID-19 with high accuracy and sensitivity. This approach is highly effective and can facilitate the early diagnosis of COVID-19 without the use of EUA reagents in the Republic of Korea.