实时荧光定量RT-PCR检测小反刍兽疫病毒方法的建立  

A SYBR GreenⅠ Based on Real-time Quantitative RT-PCR Assay for Specific Detection of Peste des Petits Ruminants Virus

作　　者：赵玲娜[1] 金红岩[1] 梁琳[1] 李刚[1] ZHAO Ling-na JIN Hong-yan LIANG Lin LI Gang(Beining Scientific Observation and Ezperiment Station for Veterinary Drug and Veterinary Biotechnology of Ministry of Agriculture, State Key Laboratory of Animal Nutrition, Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricutural Sciences, Beijing 100193, China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,动物营养学国家重点实验室,农业部兽用药物与兽医生物技术北京科学观测试验站,北京100193

出　　处：《中国畜牧兽医》2016年第11期2844-2851,共8页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(31172342);国家科技支撑计划(2013BAD12B05);转基因重大专项(2014ZX0801203B)

摘　　要：本试验旨在建立一种快速、灵敏的诊断小反刍兽疫的方法。本研究通过RT-PCR方法扩增小反刍兽疫病毒N基因,连接到pMD19-T克隆载体上,构建质粒标准品。根据GenBank中中国流行毒株及Nigeria 75/1疫苗株N基因保守序列设计引物,利用SYBR GreenⅠ法进行实时荧光定量PCR,建立标准曲线,并进行特异性试验、敏感性试验和重复性试验。结果表明,在2.82×100-2.82×10^7拷贝/μL范围内,Ct值与质粒拷贝数对数值呈良好的线性关系,标准曲线线性关系R2值为0.992;其他病毒无特异性扩增曲线,特异性良好;批内变异系数为0.27%-2.77%,批间变异系数为0.41%-3.39%,重复性较好;检测灵敏度可达2.82拷贝/μL,是普通PCR的1 000倍。用该方法对12份cDNA样品进行检测,9份为阳性,3份为阴性,而普通PCR检测,7份为阳性,5份为阴性,说明本方法比普通PCR灵敏度高。本检测方法的建立对快速、灵敏诊断小反刍兽疫,防止疫情的扩散具有重要意义。The study was aimed to establish a rapid and sensitive diagnostic method for the prevention and control of peste des petits ruminants.In this study,a fragment of PPRV N gene was amplified and cloned into pMD19-T cloning vector.Real-time quantitative PCR assay was performed using SYBR premix Ex Taq.The standard curve was plotted and the specificity,sensitivity and reproducibility of the assay were assessed.The generated standard showed linearity over the entire range from 2.82×100to 2.82×10^7 copies/μL with a linear correlation（R2）of 0.992.The specificity of the assay showed that other viruses failed to show an amplification signal.The coefficient of variation（CV）values for intra-and inter-assay variability were low,ranging from0.27%-2.77% and 0.41%-3.39%,respectively.The lower detection limit,based on plasmid copy number,achieved was 2.82copies/μL and was 1 000 times more sensitive than conventional PCR assay.cDNA of 12 samples were tested using this method,9were positive,and 3were negative.The samples were also tested using conventional PCR,7were positive and 5were negative,proving that the two-step SYBR Green Ⅰ based Real-time quantitative RT-PCR assay reported here were more sensitive than conventional PCR.The establishment of this detection method is of great significance to rapid and sensitive diagnosis of peste des petits ruminants and preventing the spread of peste des petits ruminants.

关 键 词：小反刍兽疫病毒 普通RT-PCR 实时荧光定量RT-PCR SYBR GreenⅠ 

分 类 号：S858.26[农业科学—临床兽医学]