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Development and Application of an RT_RAA Rapid Detection Method for Middle East Respiratory Syndrome Coronavirus—Ningbo National Travel & Ningbo Inspection and Quarantine
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Abstract: A nucleic acid detection method for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) based on recombinase-mediated amplification (RAA) has been established. In this study, we designed and synthesized RAA-specific primers and probes targeting the MERS-CoV genome, and prepared a positive control consisting of MERS-CoV pseudovirus particles. Through a series of optimization experiments, we developed a rapid, sensitive, and specific RT-RAA assay for MERS-CoV. The performance of the RT-RAA assay was compared with that of a fluorescence quantitative RT-PCR assay. Furthermore, clinical validation was conducted using pharyngeal swab samples from the first Chinese case imported from South Korea, as well as other respiratory virus samples and inactivated MERS-CoV proficiency testing samples provided by QC-MD in the UK. The results showed that the sensitivity of the established RT-RAA assay for detecting MERS-CoV was 10 copies, which is higher than the sensitivity of the fluorescence RT-PCR assay previously developed in our laboratory (100 copies). Moreover, the detection time (48–13.6 minutes) was significantly shorter than that of fluorescence RT-PCR (90 minutes). The RT-RAA assay yielded positive results when used to detect MERS-CoV pseudovirus particles, while all eight other control respiratory viruses tested were negative. Additionally, the assay’s results from clinical positive samples were consistent with the actual clinical outcomes. The RT-RAA assay for MERS-CoV established in this study is highly sensitive, specific, and rapid, making it suitable for rapid on-site diagnosis and epidemiological investigation of MERS-CoV infections.
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