基于NP基因的新城疫病毒Class I实时荧光定量RT-PCR方法的建立及验证  

Development and validation of a real-time fluorescent quantitative RT-PCR based on NP gene of Newcastle disease virus( NDV) Class I

作　　者：曹军平[1,2] 王晓泉[1] 程汉[2] 刘晓文[1] 刘秀梵[1] 

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏农牧科技职业学院,江苏泰州225300

出　　处：《江苏农业学报》2015年第6期1389-1394,共6页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金重点项目(30630048);国家科技支撑计划项目(2006BAD06A03)

摘　　要：新城疫病毒是影响养禽业健康发展的主要病原之一。新城疫病毒只有1个血清型,但根据亲源关系可以划分为Class I和ClassⅡ两大类。为快速定量检测Class I新城疫病毒,根据其NP基因的保守序列,设计合成1对引物和Taq Man探针,以Class I新城疫病毒JS-18-05株NP基因阳性重组质粒为标准品模板,建立荧光定量PCR标准曲线,首次建立了一种敏感、特异、重复性好的快速检测新城疫病毒Class I核酸载量的Taq Man荧光定量RT-PCR方法。该方法在102~108拷贝范围内具有良好的线性关系,可检测到初始模板中1μl 10拷贝的病毒核酸,与传统的病毒分离方法具有相近的敏感性,且这两种方法对33株Class I、ClassⅡ新城疫病毒分离株尿囊液样品检测结果符合率为100%。Newcastle disease ( ND) is one of the most serious poultry diseases. Phylogenetic analysis has revealed Newcastle disease virus ( NDV) strains include two distinct classes ( Class I and ClassⅡ) within a single serotype. To de?velop a quick method to detect NDV Class I, a pair of primers and a TaqMan probe were designed and synthesized accord?ing to NP gene conservative sequence of Newcastle disease virus Class I. The positive recombinant plasmid cloned with NP gene of JS?18?05 strain isolated from duck was used as a positive quantitative template to establish a standard curve. Then a rea1?time fluorescent quantitative RT?PCR assay was established. The method had a good linear relationship within the con?centration of 102 to 108 copies of NDV, with which 1μl 10 copy of the virus nucleic acid could be detected in the initial template. Compared with traditional methods, the assay esstablished in this study show the same results in detecting 33 NDV isolates.

关 键 词：新城疫病毒Class I 核衣壳蛋白基因 荧光定量RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]