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

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.

A Case Report: Genetically Distinct Severe Acute Respiratory Syndrome Coronavirus-2 Variant Causing Reinfection

“…RT-PCR targeted the RdRp (ORF1ab) and N genes as per the protocol described by the Chinese Center for Disease Control and Prevention (China CDC) and recommended by the World Health Organization (WHO) ( CDC, 2020 ; World Health Organization [WHO], 2020b ). This RT-PCR method has a sensitivity of 98.2% and specificity of 100% compared to different real-time RT-PCR assays ( Vogels et al, 2020 ; Chung et al, 2021 ). The patient did not require hospitalization, and after 7 days, his symptoms went away, though he experienced fatigue for two more weeks.…”

Surface-enhanced Raman scattering-based immunoassay for severe acute respiratory syndrome coronavirus 2

“…According to the previous results, the cyclic threshold (C t ) values ​​of COVID-19 patients are mainly distributed in the range of 18.12–32.23 (mean of 27.16) ( Azzi et al, 2020 ). Considering the relationship between C t value and viral concentration (PFU/mL), the COVID-19 positive samples may include about 3000 PFU/mL of the virus ( Chung et al, 2021 ). Because the present method can detect SARS-CoV-2 at lower concentrations than 3000 PFU/mL, we think the method has the potential for the diagnosis of COVID-19 patients.…”

SARS-CoV-2 in wastewater: From detection to evaluation

“…RT-PCR and isothermal nucleic acid amplification are two well-developed amplification methods diagnostic tools. RT-PCR tests, as one of the most used SARS-CoV-2 detection methods [ 86 , 87 ], will be further described in Section 2.3. Despite the wide adaptation of PCR, PCR machines are costly, and its thermal cycling process is time consuming.…”