实时荧光定量RT-PCR检测禽白血病病毒方法的建立与应用  

Development of real-time quantitative RT-PCR assay for detection of ALV in chickens

作　　者：张太翔[1] 凌宗帅[2] 史玉颖[3] 高小博 田国宁[1] 张金玲[1] 韩亮[1] 张丽萍[1] 

机构地区：[1]潍坊出入境检验检疫局,山东潍坊261041 [2]济南出入境检验检疫局,山东济南250010 [3]山东省农业科学院家禽研究所,山东济南250023 [4]北京盈九思科技发展有限公司,北京朝阳100029

出　　处：《中国兽医杂志》2010年第6期3-6,共4页Chinese Journal of Veterinary Medicine

基　　金：山东出入境检验检疫局科研课题(SK200896)

摘　　要：建立了TaqMan实时荧光定量RT-PCR方法检测禽白血病病毒（ALV）。选取ALV病毒的LTR序列设计引物和探针,以梯度稀释的含有ALV目的扩增片段的质粒作为标准品,进行定量PCR反应以确定检测灵敏度。阳性标准品在3.0×102~3.0×107个拷贝共6个数量级的范围内,定量PCR反应有＂S＂型扩增曲线,检测灵敏度最低为30个拷贝。根据病毒拷贝数与定量反应Ct值的关系,绘制了标准曲线。该方法具有特异性,对新城疫病毒、禽流感病毒、传染性支气管炎病毒、传染性囊病病毒、鸡传染性贫血病毒和马立克病病毒核酸都没有扩增反应。实时定量PCR检测ALV的方法,灵敏度高,特异性好,可以进行定量分析,在禽病的快速检测上具有重要意义。A real-time quantitative RT-PCR assay was developed for the detection of avian leukosis virus（ALV）.Primers and probe were designed based on the nucleocapsid gene of ALV by Primer Express 2.0 software.The plasmid containing the target sequence was constructed to detect the sensitivity and prepare the standard curve.The real-time RT-PCR assay had a detection limit of 30 copies,with a dynamic range of detection between 3×107~3×102 copies.The standard curve was prepared based on the linear relationship between the amount of plasmid DNA and cycle threshold（Ct）.The primers and probe were specific for ALV and did not react with either collected samples were detected with the real-time RT-PCR assay and three positive samples were used for quantitative analysis.The real-time RT-PCR that we developed with high sensitivity,specificity and accuracy is considered to be a powerful tool for the rapid detection and quantification of ALV in avian.

关 键 词：禽白血病 实时荧光定量RT-PCR TAQMAN探针 

分 类 号：S852.659.3[农业科学—基础兽医学]