关键词: 鸽Ⅰ型副黏病毒；鸽圆环病毒；鸽轮状病毒；鸽腺病毒；四重TaqMan qPCR

Abstract: To develop a quadruplex TaqMan real ? time quantitative PCR assay for the accurate and rapid detection of Pigeon Paramyxovirus Type 1（PPMV?1），Pigeon Circovirus（PiCV），Pigeon Rotavirus（PiRV），and Pigeon Adenovirus（PiAdV），specific primers and probes were designed based on the conserved gene sequences of the four viruses．By optimizing primer/ probe concentrations and amplification conditions，a quadruplex detection system was established and subsequently evaluated for standard curve linearity，specificity，sensitivity，repeatability，and clinical applicability were evaluated． The standard curves for all four targets exhibited excellent linearity，with R2 values all greater than 0.99；no cross ? amplification occurred with nucleic acids of other common pigeon viruses， indicating good specificity；the minimum detectable limits for standard plasmids PPMV?1，PiCV，PiRV，and PiAdV were 1×101，1×101，1×101，and 1×102 copies/μL，respectively；intra? and inter?assay coefficient of variation（CV）of CT values for both intra ? and inter ? assay experiments was less than 2% ，showing low fluctuation and good repeatability. To evaluate the method's applicability and reliability，115 pigeon clinical samples were tested，and the positive detection rates for PPMV?1，PiCV，PiRV，and PiAdV were 20.87%，60.87%，15.65%，and 41.73%， respectively，suggesting a high proportion of multi ? pathogen infections in clinical samples. In summary，the quadruplex TaqMan real?time quantitative PCR method established in this study demonstrates high sensitivity，strong specificity，and good repeatability，providing a reliable technical tool for the rapid screening and diagnosis of common pigeon viruses.

Key words: Pigeon Paramyxovirus Type 1；Pigeon Circovirus；Pigeon Rotavirus；Pigeon Adenovirus；Quadruplex TaqMan qPCR