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

摘要： 为构建精准、快速的检测鸽Ⅰ型副黏病毒（PPMV?1）、鸽圆环病毒（PiCV）、鸽轮状病 毒（PiRV）和鸽腺病毒（PiAdV）的四重TaqMan实时荧光定量PCR检测方法，根据四种病毒保守 基因序列设计特异性引物和探针，通过优化引物探针浓度及扩增条件，构建四重检测体系，并 对其标准曲线、特异性、敏感性、重复性及临床适用性进行评价。结果显示，成功建立标准曲 线，R2 均大于0.99；与其他常见鸽病毒核酸均无交叉扩增，具有良好的特异性；对标准质粒 PPMV?1、PiCV、PiRV和PiAdV的最低检出限分别为1×101、1×101、1×101和1×102 copies/μL；批内 及批间实验CT值变异系数均小于2%，各组波动性较小，重复性良好。为评估该方法的适用性 和可靠性，对115份临床鸽样品进行检测，PPMV?1、PiCV、PiRV和PiAdV的阳性检出率分别为 20.87%、60.87%、15.65%和41.73%，提示临床样品中存在较高比例的多病原感染现象。综上， 该试验建立的四重TaqMan实时荧光定量PCR方法具有敏感性高、特异性强和重复性好等优 势，为鸽常见病毒的快速筛查及诊断提供技术支持。

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