实时荧光定量RT-PCR检测小反刍兽疫病毒方法的建立

doi:10.16431/j.cnki.1671-7236.2016.11.006

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (11): 2844-2851.doi: 10.16431/j.cnki.1671-7236.2016.11.006

实时荧光定量RT-PCR检测小反刍兽疫病毒方法的建立

赵玲娜, 金红岩, 梁琳, 李刚

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

收稿日期:2016-04-14 出版日期:2016-11-20 发布日期:2016-11-18
通讯作者: 李刚 E-mail:ligang03@caas.cn
作者简介:赵玲娜(1989-),女,河北保定人,硕士生,研究方向:预防兽医学,E-mail:zhaolingna1011@163.com
基金资助:
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）

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

ZHAO Ling-na, JIN Hong-yan, LIANG Lin, LI Gang

Beining Scientific Observation and Experiment 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

Received:2016-04-14 Online:2016-11-20 Published:2016-11-18

摘要/Abstract

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 Green Ⅰ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2.82×10⁰~2.82×10⁷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。

关键词: 小反刍兽疫病毒; 普通RT-PCR; 实时荧光定量RT-PCR; SYBR Green Ⅰ

Abstract:

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 2.82×10⁰ to 2.82×10⁷ 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 from 0.27%~2.77% and 0.41%~3.39%,respectively.The lower detection limit,based on plasmid copy number,achieved was 2.82 copies/μL and was 1 000 times more sensitive than conventional PCR assay.cDNA of 12 samples were tested using this method,9 were positive,and 3 were negative.The samples were also tested using conventional PCR,7 were positive and 5 were 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.

Key words: peste des petits ruminants virus; conventional RT-PCR; Real-time quantitative RT-PCR; SYBR Green Ⅰ

中图分类号:

S858.26

赵玲娜, 金红岩, 梁琳, 李刚. 实时荧光定量RT-PCR检测小反刍兽疫病毒方法的建立[J]. 《中国畜牧兽医》, 2016, 43(11): 2844-2851.

ZHAO Ling-na, JIN Hong-yan, LIANG Lin, LI Gang. A SYBR Green Ⅰ Based on Real-time Quantitative RT-PCR Assay for Specific Detection of Peste des Petits Ruminants Virus[J]. , 2016, 43(11): 2844-2851.

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实时荧光定量RT-PCR检测小反刍兽疫病毒方法的建立

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

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