Diagnostic performance and clinical feasibility of a novel one-step RT-qPCR assay for simultaneous detection of multiple severe acute respiratory syndrome coronaviruses


Le Tran Bac,Kim Hye Kwon,Ahn Min-Ju,Zanin Mark,Lo Van Thi,Ling Shiman,Jiang Zhanpeng,Kang Jung-Ah,Bae Pan Kee,Kim Yeon-Sook,Kim Seungtaek,Wong Sook-San,Jeong Dae Gwin,Yoon Sun-WooORCID


AbstractCoronavirus disease 2019 (COVID-19) is an acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Other coronaviruses (CoVs) can also infect humans, although the majority cause only mild respiratory symptoms. Because early diagnosis of SARS-CoV-2 is critical for preventing further transmission events and improving clinical outcomes, it is important to be able to distinguish SARS-CoV-2 from other SARS-related CoVs in respiratory samples. Therefore, we developed and evaluated a novel reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the genes encoding the spike (S) and membrane (M) proteins to enable the rapid identification of SARS-CoV-2, including several new circulating variants and other emerging SARS-like CoVs. By analysis of in vitro-transcribed mRNA, we established multiplex RT-qPCR assays capable of detecting 5 × 10° copies/reaction. Using RNA extracted from cell culture supernatants, our multiple simultaneous SARS-CoV-2 assays had a limit of detection of 1 × 10° TCID50/mL and showed no cross-reaction with human CoVs or other respiratory viruses. We also validated our method using human clinical samples from patients with COVID-19 and healthy individuals, including nasal swab and sputum samples. This novel one-step multiplex RT-qPCR assay can be used to improve the laboratory diagnosis of human-pathogenic CoVs, including SARS-CoV-2, and may be useful for the identification of other SARS-like CoVs of zoonotic origin.


National Research Foundation of Korea

Ministry of Science, ICT and Future Planning


Springer Science and Business Media LLC


Virology,General Medicine








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