Abstract
Background: An outbreak of novel coronavirus (SARS-CoV-2)-associated respiratory infectious diseases (COVID-19) emerged in 2019 and has spread rapidly in humans around the world. The demonstration of in vitro infectiousness of respiratory specimens is an informative surrogate for SARS-CoV-2 transmission from patients with COVID-19; accordingly, viral isolation assays in cell culture are an important aspect of laboratory diagnostics for COVID-19.
Methods: We developed a simple and rapid protocol for isolating SARS-CoV-2 from respiratory specimens using VeroE6/TMPRSS2 cells, a cell line that is highly susceptible to the virus. We also investigated a correlation between isolation of SARS-CoV-2 and viral load detected by real-time RT-PCR (rRT-PCR) using N2 primer/probe set that has been developed for testing of COVID-19 in Japan.
Results: The SARS-CoV-2 isolation protocol did not require blind passage of inoculated cells and yielded the results of viral isolation within 7 days after inoculation. Specimens with cycle threshold (Ct) values of <20.2, determined by rRT-PCR, were predicted to be isolation-positive. On the other hand, 6.9% of specimens with Ct values >35 were virus isolation-positive, indicating that low viral loads (high Ct values) in upper respiratory specimens do not always indicate no risk of containing transmissible virus.
Conclusion: In combination with rRT-PCR, the SARS-CoV-2 isolation protocol provides a means for assessing the potential risk of transmissible virus in upper respiratory specimens.
Keywords: COVID-19; viral infection.
【저자키워드】 COVID-19, viral infection, 【초록키워드】 SARS-CoV-2, viral infection, protocol, Human, in vitro, virus, diagnostics, Novel coronavirus, Laboratory, Spread, rRT-PCR, SARS-CoV-2 transmission, Viral, Viral load, virus isolation, cells, cycle threshold, outbreak, inoculation, infectiousness, Cell culture, Patient, Isolation, Japan, respiratory, real-time RT-PCR, correlation, specimens, Combination, respiratory specimens, Ct values, determined by, blind passage, no risk, potential risk, specimen, demonstration, respiratory infectious disease, cell line, blind, susceptible, Cell, upper respiratory, respiratory specimen, predicted, inoculated, investigated, provide, the SARS-CoV-2, 【제목키워드】 SARS-CoV-2, assessment, COVID-19 patient, specimen, Cell, upper respiratory,