South Korea, and attempts have been made to isolate the pathogen from these patients. Methods: Upper and lower respiratory tract secretion samples from putative patients with COVID-19 were inoculated onto cells to isolate the virus. Full genome sequencing and electron microscopy were used to identify the virus. Results: The virus replicated in Vero cells and cytopathic effects were observed. Full genome sequencing showed that the virus genome exhibited sequence homology of more than 99.9% with SARS-CoV-2 which was isolated from patients from other countries, for instance China. Sequence homology of SARS-CoV-2 with SARS-CoV, and MERS-CoV was 77.5% and 50%, respectively. Coronavirus-specific morphology was observed by electron microscopy in virus-infected Vero cells. Conclusion: SARS-CoV-2 was isolated from putative patients with unexplained pneumonia and intermittent coughing and fever. The isolated virus was named BetaCoV/Korea/KCDC03/2020.

Coronavirus Disease-19 (COVID-19) is a respiratory infection characterized by the main symptoms of pneumonia and fever. It is caused by the novel coronavirus severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2), which is known to spread via respiratory droplets. We aimed to determine the rate and likelihood of SARS-CoV-2 transmission from COVID-19 patients through nonrespiratory routes. Methods: Serum, urine, and stool samples were collected from 74 hospitalized patients diagnosed with COVID-19 based on the detection of SARS-CoV-2 in respiratory samples. The SARS-CoV-2 RNA genome was extracted from each specimen and real-time reverse transcription polymerase chain reaction performed. CaCo-2 cells were inoculated with the specimens containing the SARS-COV-2 genome, and subcultured for virus isolation. After culturing, viral replication in the cell supernatant was assessed. Results: Of the samples collected from 74 COVID-19 patients, SARS-CoV-2 was detected in 15 serum, urine, or stool samples. The virus detection rate in the serum, urine, and stool samples were 2.8% (9/323), 0.8% (2/247), and 10.1% (13/129), and the mean viral load was 1,210 ± 1,861, 79 ± 30, and 3,176 ± 7,208 copy/µL, respectively. However, the SARS-CoV-2 was not isolated by the culture method from the samples that tested positive for the SARS-CoV-2 gene. Conclusion: While the virus remained detectable in the respiratory samples of COVID-19 patients for several days after hospitalization, its detection in the serum, urine, and stool samples was intermittent. Since the virus could not be isolated from the SARS-COV-2-positive samples, the risk of viral transmission via stool and urine is expected to be low.

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.

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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.

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