Validation of real-time RT-PCR for detection of SARS-CoV-2 in the early stages of the COVID-19 outbreak in the Republic of Korea

Yoon-Seok Chung,Nam-Joo Lee,Jeong-Min Kim,Heui Man Kim,Myung-Guk Han,Ye Eun Park,Sang Hee Woo,Hye Jun Jo

doi:10.1038/s41598-021-94196-3

Abstract

A real-time reverse transcription polymerase chain reaction (RT-qPCR) assay that does not require Emergency Use Authorization (EUA) reagents was tested and validated for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the early stages of the outbreak of coronavirus disease 2019 (COVID-19) in the Republic of Korea. 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 the RT-qPCR assay for SARS-CoV-2 detection and compared its performance with that of several EUA-approved kits. Our RT-qPCR assay was highly specific for SARS-CoV-2 as demonstrated by not amplifying 13 other viruses that cause respiratory diseases. The assay showed high linearity using a viral isolate from a patient with known COVID-19 as well as plasmids containing target SARS-CoV-2 genes as templates. The assay showed good repeatability and reproducibility with a coefficient of variation of 3%, and a SARS-CoV-2 limit of detection of 1 PFU/mL. The RT-qPCR-based assay is highly effective and can facilitate the early diagnosis of COVID-19 without the use of EUA-approved kits or reagents in the Republic of Korea.

Highlights

Coronavirus disease 2019, officially named COVID-19 by the World Health Organization (WHO) is a severe acute respiratory syndrome (SARS) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARSCoV-2)
To examine the responsivity of the assay, RT-qPCR was performed using tenfold serially diluted RNA extracted from a lower respiratory tract sample of the first patient who tested positive for COVID-19 in Republic of Korea
Viral RNA specific to human coronaviruses 229E, NL63, OC43, and HKU1; SARS-CoV; MERS-CoV; influenza virus; adenovirus; rhinovirus; parainfluenza virus; respiratory syncytial virus; metapneumovirus; and bocavirus was not detected in the RT-qPCR specificity assay using primers targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and E genes (Table 3)

Summary

Introduction

Coronavirus disease 2019, officially named COVID-19 by the World Health Organization (WHO) is a severe acute respiratory syndrome (SARS) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARSCoV-2). SARS-CoV-2 belongs to the subfamily Orthocoronavirinae, which is a member of the family Coronaviridae[7] This beta coronavirus has a 30-kb genome, sharing 96% sequence identity with the bat coronavirus RaTG13, 88% with bat coronaviruses ZC45 and ZXC21, 80% with SARS-CoV, and 50% with MERS-CoV8. COVID-19 can be diagnosed in the laboratory by detecting SARS-CoV-2 genes in clinical samples collected from suspected patients, Scientific Reports | (2021) 11:14817. Published protocols differ based on the target genes, primer and probe sequences, mixture composition, amplification conditions, sensitivity, and the requirement for Emergency Use Authorization (EUA)-approved reagents. This was especially true during the early outbreak of COVID-19 in Korea when most protocols required the use of EUA-approved specific reagents for RT-qPCR, some of which were not available in Korea. We compared the accuracy of our assay results with those obtained using five different COVID-19 EUA-approved kits and respiratory samples from patients with and without suspected COVID-19

Methods

Results

Conclusion