Development of a universal real-time RT-PCR assay for detection of pan-SARS-coronaviruses with an RNA-based internal control

Beibei Yu; Changping Xu; Shiwang Huang; Jun Ni; Jiancang Zhou; Yuting Zhang; Maomao Wu; Jun Zhang; Lei Fang

doi:10.3389/fmicb.2023.1181097

Development of a universal real-time RT-PCR assay for detection of pan-SARS-coronaviruses with an RNA-based internal control

Front Microbiol. 2023 May 18:14:1181097. doi: 10.3389/fmicb.2023.1181097. eCollection 2023.

Authors

Beibei Yu^#^{1

2}, Changping Xu^#³, Shiwang Huang^#⁴, Jun Ni^{5

6}, Jiancang Zhou^{5

6}, Yuting Zhang³, Maomao Wu⁷, Jun Zhang^{1

2}, Lei Fang^{5

6}

Affiliations

¹ Department of Clinical Laboratory, Sir Run Run Shaw Hospital, Zhejiang University College of Medicine, Hangzhou, China.
² Key Laboratory of Precision Medicine in Diagnosis and Monitoring Research of Zhejiang Province, Hangzhou, China.
³ Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, China.
⁴ Shangcheng District Center for Disease Control and Prevention, Hangzhou, China.
⁵ Department of Critical Care Medicine, Sir Run Run Shaw Hospital, Zhejiang University College of Medicine, Hangzhou, China.
⁶ Key Laboratory of Microbial Technology and Bioinformatics of Zhejiang Province, Hangzhou, China.
⁷ Wenzhou Center for Disease Control and Prevention, Wenzhou, China.

^# Contributed equally.

Abstract

The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for rapid diagnostic assays to prompt intensified virological monitoring both in human and wild animal populations. To date, there are no clinical validated assays for pan-SARS-coronavirus (pan-SARS-CoV) detection. Here, we suggest an innovative primer design strategy for the diagnosis of pan-SARS-CoVs targeting the envelope (E) gene using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, we developed a new primer-probe set targeting human β₂-microglobulin (B2M) as an RNA-based internal control for process efficacy. The universal RT-qPCR assay demonstrated no false-positive amplifications with other human coronaviruses or 20 common respiratory viruses, and its limit of detection (LOD) was 159.16 copies/ml at 95% detection probability. In clinical validation, the assay delivered 100% sensitive results in the detection of SARS-CoV-2-positive oropharyngeal samples (n = 120), including three variants of concern (Wuhan, Delta, and Omicron). Taken together, this universal RT-qPCR assay provides a highly sensitive, robust, and rapid detection of SARS-CoV-1, SARS-CoV-2, and animal-derived SARS-related CoVs.

Keywords: COVID-19; SARS; SARS-CoV-2; animal coronaviruses; molecular diagnostics; real-time RT-PCR.

Grants and funding

This study was supported by the Zhejiang Provincial Basic Commonweal Research Project (LGC22H200002), Zhejiang Provincial Medical Science and Technology Platform Project (2021KY122), National Health Commission Scientific Research Project (WKJ-ZJ-2112), China Postdoctoral Science Foundation (2020T130104ZX), and Zhejiang Postdoctoral Science Foundation (ZJ2020027).