基因Ⅶ型新城疫病毒荧光定量RT-PCR检测方法的建立  

Development of Real-time Fluorescent Quantitative RT-PCR Assay for Detection of Genotype Ⅶ Newcastle Disease Virus (NDV)

作　　者：孙军峰[1,2] 刘华雷[1] 王志亮[1] 赵云玲[1] 郑东霞[1] 张维[1] 刘文华[1] 冯莉莉[1] 常娓娓[1] 

机构地区：[1]中国动物卫生与流行病学中心,青岛266032 [2]青岛农业大学,青岛266109

出　　处：《畜牧兽医学报》2010年第11期1447-1452,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：青岛市科技发展计划项目(07-2-3-5-jch)

摘　　要：根据基因ⅤⅡ型新城疫病毒F基因裂解位点特征设计一对引物和TaqMan探针,建立一种敏感、特异、重复性良好的基因ⅤⅡ型毒株荧光定量RT-PCR鉴别检测方法。以体外转录法制备的cRNA标准品为标准阳性模板,构建标准曲线,并进行各项指标的检测。结果表明,建立的方法特异性良好,最低可检测到102拷贝·μL-1的模板RNA,敏感性比普通RT-PCR高100倍。本试验建立了基因ⅤⅡ型新城疫病毒荧光定量RT-PCR检测方法,且该方法能够鉴别检测常规方法不能区分的新城疫病毒基因ⅤⅡ型强毒株,表明本研究建立的方法可用于此种基因型毒株的快速鉴别检测。In order to establish a differential diagnosis Real-time RT-PCR with highly sensitivity,specificity and repeatability,a pair of primers and TaqMan probe were designed according to characteristics of cleavage site sequences of F gene of genotype Ⅶ Newcastle disease virus strains in this study.The standard curve was established by using cRNA standard prepared in vitro transcription as positive template,the related indexes were tested.The results showed that this assay has a good effect of differential detection of genotype Ⅶ NDV;102 copies·μL-1 of template RNA could be detected at least.The sensitive of this assay was 100 times higher than conventional RT-PCR.The real-time fluorescent quantitative RT-PCR for detection of genotype Ⅶ Newcastle disease virus was established.This assay could be used as differential diagnosis method of genotype Ⅶ NDV strains,which couldn’t be identified by conventional methods.Our results indicated that the method established in this study can be used as rapid identification assay for the detection of genotype Ⅶ NDVs.

关 键 词：新城疫病毒 基因Ⅶ型 荧光定量RT-PCR 

分 类 号：S852.659.5[农业科学—基础兽医学]