鸽圆环病毒荧光定量PCR检测方法的建立及应用  

Development and Application of a Real-time Fluorescence Quantitative PCR Detection Method for Pigeon Circovirus

作　　者：王星月 杨志远[2] 林健[2] 张紫萱 周雨婷 程慧敏 刘月焕[2] 胡格[1] WANG Xingyue;YANG Zhiyuan;LIN Jian;ZHANG Zixuan;ZHOU Yuting;CHENG Huimin;LIU Yuehuan;HU Ge(College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206,China;Institute of Animal Husbandry and Veterinary Medicine,Beijing Academy of Agricultural and Forestry Sciences,Beijing 100097,China)

机构地区：[1]北京农学院动物科学技术学院,北京102206 [2]北京市农林科学院畜牧兽医研究所,北京100097

出　　处：《畜牧兽医学报》2025年第1期335-342,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：现代农业产业技术体系北京市家禽创新团队(BAIC06-2024);北京市农林科学院科技创新能力建设专项(KJCX20220422)。

摘　　要：为实现对鸽圆环病毒(pigeon circovirus, PiCV)的快速检测,建立了针对Cap基因的PiCV荧光定量PCR检测方法,并应用该方法进行PiCV分子流行病学调查。本研究针对PiCV核衣壳蛋白Cap基因的保守序列设计一对特异性引物,并通过优化反应条件,成功建立了荧光定量PCR检测方法。此外,还对该方法的灵敏度、特异性、重复性进行了全面评估。利用建立的荧光定量PCR方法对华北和西北部分地区147份临床样本进行检测,并对阳性代表样本的Cap全长序列进行遗传演化分析。结果表明,所建立的荧光定量PCR方法在1×10^(4)至1×10^(8)拷贝·μL^(-1)范围内显示出良好的线性关系,最低检测限达到1×10^(1)拷贝·μL^(-1),显著高于传统PCR方法。此方法具有良好的重复性,且不与其他常见鸽病病原产生交叉反应。采用本试验方法检测的147份华北和西北部分地区肉鸽和信鸽临床样本PiCV阳性率为85.0%,阳性样本的Cap全长序列分析及遗传演化分析结果显示,6株分离株相似性在89.8%~97.5%之间,与PiCV HeBeiTS2021株在同一个分支,亲缘关系较近。建立的PiCV荧光定量PCR方法具备灵敏、特异和稳定等优点,可用于PiCV的快速检测,同时流行病学调查结果表明,2022—2023年华北和西北部分地区PiCV感染率较高,为我国PiCV的防控研究提供数据支撑。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 1×10^(4) to 1×10^(8) copies·μL^(-1),with a minimum detection limit as low as 1×10^(1) 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.

关 键 词：鸽圆环病毒 Cap基因 荧光定量PCR 分子流行病学 遗传演化分析 

分 类 号：S852.659.2[农业科学—基础兽医学]