Abstract:

The aim of this study was to establish a rapid diagnostic method of pigeon circovirus (PiCV), a real-time quantitative PCR based on Cap gene was established and a molecular epidemiological investigation on PiCV was conducted. Specific primers targeting the conserved sequence of the Cap gene of PiCV were designed and synthesized. The sensitivity, specificity, and repeatability of the developed quantitative PCR method (qPCR) were evaluated. One hundred and forty-seven clinical samples from Northern and Northwest China were detected by the qPCR, followed by genome sequencing and phylogenetic analysis on whole length of Cap gene of isolates from different regions. The established qPCR method showed a good linear response for PiCV standards ranging from 1×10⁴ to 1×10⁸ copies·μL^-1, with a minimum detection limit as low as 1×10¹ copies·μL^-1, substantially surpassing conventional PCR. The intra-assay and inter-assay demonstrated a good repeatability. Importantly, there was no cross-reactivity with other pigeon pathogens. Detection results of 147 clinical samples collected from Northern and Northwest China by this method showed that the positive rate for PiCV was 85.0%. Sequencing results and phylogenetic analysis revealed that Cap gene of six isolates shared homology within the range of 89.8% to 97.5%, and were closely related to PiCV HeBeiTS2021 isolate. The established real-time quantitative PCR method for PiCV demonstrated sensitivity, specificity and repeatability, making it available for rapid detection of PiCV. Epidemiological investigation results indicated a higher PiCV infection rate in Northern and Northwest China during 2022-2023, providing valuable data for prevention and control of PiCV.

Key words: pigeon circovirus, Cap gene, real-time fluorescence quantitative PCR, molecular epidemiology, phylogenetic analysis