Establishment of a TaqMan MGB probe real-time PCR detection method for pigeon circovirus and its whole genome analysis

<bold>Objective</bold> Pigeon circovirus（PiCV）infection is commonly associated with impaired immune systems in pigeons.By suppressing the host’s immune response，it creates conditions for other pathogens to invade.Mixed infections can cause symptoms similar to other avian diseases，such as respiratory issues，diarrhea，and reduced appetite，leading to economic losses in pigeon farming and complicating diagnosis.This study aims to develop a rapid and efficient TaqMan MGB probe-based real-time quantitative PCR（qPCR）method for PiCV detection，providing a technical tool for clinical diagnosis and epidemiological investigation. <bold>Method</bold> The conserved region of the Rep protein gene of PiCV was identified by comparing the full genomes of PiCV available in GenBank.Primers，an MGB probe，and a plasmid standard（pMD19-T-PiCV）were designed based on this conserved region.The qPCR method was optimized，and a standard curve was generated to evaluate the specificity，sensitivity，and repeatability of the method.The developed qPCR method was used to test clinical samples，and the full genomes of positive samples were sequenced for homology and phylogenetic analysis. <bold>Result</bold> The standard curve of the PiCV TaqMan MGB probe qPCR method exhibited a linear relationship with the equation <italic>y</italic>=-3.376 0<italic>x</italic>+39.260 0，a correlation coefficient <italic>R</italic>² of 0.996 4，and an amplification efficiency of 97.79%.The method showed no cross-reactivity with other common pigeon viruses（e.g.，pigeon adenovirus and pigeon herpesvirus），demonstrating high specificity.The detection limit was as low as 3.061 copies/μL，which is 1 000 times more sensitive than conventional PCR.Intra- and inter-assay repeatability tests showed coefficient of variation values below 1.500%，indicating excellent stability.Clinical testing of 40 pigeon samples revealed a positivity rate of 32.50%，higher than the 20.00% positivity rate achieved by conventional PCR.Homology and phylogenetic analyses of the PiCV full genome，Rep protein gene，and Cap protein gene from clinical samples showed 95.14%-96.96% similarity with reference strains，with the full genome and Cap protein gene clustering closely with strains MN920392.1 and PP405070.1. <bold>Conclusion</bold> The developed PiCV TaqMan MGB probe qPCR method features a well-defined standard curve，high specificity，sensitivity，and repeatability.It provides a rapid，efficient，and specific tool for detecting PiCV in clinical samples，offering a novel technique for early diagnosis and epidemiological investigation of pigeon circovirus.