Abstract: The present study aimed to establish a novel real-time fluorescent RT-PCR assay for detection of pigeon megrivirus (PiMeV). Specific primers were designed based on PiMeVs sequences download from the GenBank, and positive fragments (designated as PiMeV-CHN001 strain) were amplified from racing pigeon feces. Then the 3C gene of PiMeV-CHN001 strain was obtained and analyzed. Based on the 3C molecular characterization, a real-time fluorescent RT-PCR method (RT-qPCR) was developed using the specific primers. The results demonstrated the 3C gene of PiMeV-CHN001 strain had 591 bp (coding 197 amino acids), with the nucleic acid sequence homology with other wild urban pigeons (Columba livia) (MeV-B1 strain and MeV-B2 strain) was 89.5% and 92.0%, respectively. The RT-qPCR assay standard curve had the axial intercept of standard curve was 37.93 and the slope was -3.335, with a linear correlation (R2) of 1.00 and efficiency of 99.4%. The methods were specificity, no-cross amplification signal was found from other pigeon viruses (such as avian influenza virus, pigeon paramyxovirus type I, pigeon torque teno virus, pigeon adenovirus, and pigeon circovirus). Only one specific peaked with a melting temperature (Tm) was (81.69±0.22) ℃ form PiMeV-CHN001, with no primer-dimers peak represent. The lowest limit of detection concentration was 54.0 copies/μL. The intra-and inter-assay were less than 1.5% according to the repeatability test. The developed qPCR was used for PiMeV detection of 42 racing pigeon feces. 2 positive (positive rate was 4.76%) signals were found. In conclusion, we firstly confirmed the presence of PiMeV in racing pigeons in Mainland China, and the data can enrich Megrivirus host spectrum. Moreover, the developed RT-qPCR assay also lays good foundation for further PiMeV epidemiological investigation.

Key words: racing pigeon, pigeon megrivirus, 3C gene, sequence analysis, real-time fluorescent quantitative RT-PCR assay