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

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

doi:10.1007/s00705-022-05383-0

Abstract

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° 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.

Highlights

Coronaviruses (CoVs) are single-stranded, positive-sense RNA viruses belonging to the family Coronaviridae that can infect both animals and humans [1,2,3]
Primers targeting the RNA-dependent RNA polymerase (RdRp) gene for detection of severe acute respiratory syndrome (SARS)-CoV-2 cross-react with SARS-CoV RNA [11], and some researchers have found that the sensitivity of reverse transcription quantitative polymerase chain reaction (RT-qPCR) is only approximately 70% for SARS-CoV-2 diagnosis [13], which may result in false-negative results
These results suggest that, a protocol using RT-qPCR assay for SARS-CoV-2 detection using RdRp, E, and N genes has been established, alternative molecular diagnostic targets are needed to improve the sensitivity of SARS-CoV-2 virus detection

Summary

Introduction

Coronaviruses (CoVs) are single-stranded, positive-sense RNA viruses belonging to the family Coronaviridae that can infect both animals and humans [1,2,3]. Primers targeting the RdRp gene for detection of SARS-CoV-2 cross-react with SARS-CoV RNA [11], and some researchers have found that the sensitivity of RT-qPCR is only approximately 70% for SARS-CoV-2 diagnosis [13], which may result in false-negative results. These results suggest that, a protocol using RT-qPCR assay for SARS-CoV-2 detection using RdRp, E, and N genes has been established, alternative molecular diagnostic targets are needed to improve the sensitivity of SARS-CoV-2 virus detection

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Conclusion