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

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

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1181097

DOI=10.3389/fmicb.2023.1181097

ISSN=1664-302X

ABSTRACT=<p>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 (<italic>E</italic>) gene using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, we developed a new primer–probe set targeting human β<sub>2</sub>-microglobulin (<italic>B2M</italic>) 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 (<italic>n</italic> = 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.</p>