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

• Published in: Archives of virology Vol. 167; no. 3; pp. 871 - 879
• Main Authors: 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-Woo
• Format: Journal Article
• Language: English
• Published: Vienna Springer Vienna 01-03-2022; Springer Nature B.V
• Subjects: Biomedical and Life Sciences; Biomedicine; Cell culture; Clinical Laboratory Techniques; Coronaviridae; Coronaviruses; COVID-19; COVID-19 - diagnosis; Cross-reaction; Diagnosis; Disease transmission; Feasibility Studies; Humans; Infectious Diseases; Medical Microbiology; Original; Original Article; Polymerase chain reaction; Real-Time Polymerase Chain Reaction; Reverse transcription; SARS-CoV-2 - genetics; Sensitivity and Specificity; Severe acute respiratory syndrome coronavirus 2; Sputum; Virology
• ISSN: 0304-8608; 1432-8798
• DOI: 10.1007/s00705-022-05383-0

Coronavirus 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° TCID
50
/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.