鸡传染性贫血病毒TaqMan实时荧光定量PCR方法的建立及应用  

Development and application of a TaqMan real-time PCR for the detection of chicken infectious anemia virus

作　　者：孙文杰 宋亚鹏[1] 刘琳[1] 赵振超 陶明月 董松岭 高文明[1] 李新生[1] SUN Wenjie;SONG Yapeng;LIU Lin;ZHAO Zhenchao;TAO Mingyue;DONG Songling;GAO Wenming;LI Xinsheng(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;Shangqiu Center for Quality and Technical Supervision and Testing,Shangqiu,Henan 476000,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450046 [2]商丘市质量技术监督检验测试中心,河南商丘476000

出　　处：《中国兽医学报》2023年第6期1156-1161,共6页Chinese Journal of Veterinary Science

基　　金：河南省揭榜挂帅科技资助项目(211110111000)。

摘　　要：根据鸡传染性贫血病毒(chicken infectious anemia virus, CIAV) HN1415流行株基因组序列(GenBank登录号:MZ668717)设计、合成1对特异性引物及TaqMan探针,经敏感性、特异性以及批内、批间可重复性检验对该方法进行验证。结果显示:该方法灵敏,10拷贝/μL病毒即可检出;特异性强,与FAdV-4、IBDV、AIV、IBV、ALV以及NDV等临床常发禽类传染病病原均无交叉反应;重复性好,批内及批间变异系数均小于1%。结果表明,本试验成功建立了检测CIAV的TaqMan荧光定量PCR方法。用该方法对采自中国不同养鸡地区的253份疑似CIAV感染病例进行检测,阳性检出率在41.18%~62.50%之间,其中广东省的阳性检出率最低(41.18%),山东省阳性检出率最高(62.50%)。对阳性检出样品随机测序验证结果显示,本试验建立的TaqMan探针实时荧光定量PCR方法可快速、准确检出CIAV,为CIAV的快速确诊提供了重要工具。To detect chicken infectious anemia virus(CIAV),a pair of specific primers and a Taq-Man probe were designed and synthesized based on the conserved sequence of the CIAV HN1415 strain(GenBank accession No.MZ668717).The sensitivity assay showed that the minimum detection limit was 10 copies of CIAV.Specificity assay demonstrated no cross-reaction of CIAV with common clinical avian infectious diseases such as FAdV-4,IBDV,AIV,IBV,ALV,NDV,and so on.The coefficient of variation within and between batches was less than 1%,These results indicated that the TaqMan fluorescence quantitative PCR method for detecting CIAV was successfully established.Detection of 253 suspected CIAV infection cases collected from the main chicken-rearing areas in China revealed a total positive rate of 41.18%-62.50%.The lowest positive detection rate was 41.18%in Guangdong Province,while the highest positive detection rate was 62.50%in samples from Shandong Province.The results of random sequencing validation of positive samples showed that the real-time PCR method established in this study could detect CIAV quickly,accurately,and quantitatively,which provides a tool for rapid diagnosis of CIAV.

关 键 词：鸡传染性贫血病毒 TAQMAN探针 荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]