中国畜牧兽医2016，Vol.43Issue(11)：2844-2851,8.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
A SYBR Green Ⅰ Based on Real-time Quantitative RT-PCR Assay for Specific Detection of Peste des Petits Ruminants Virus
摘要
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.关键词
小反刍兽疫病毒/普通RT-PCR/实时荧光定量RT-PCR/SYBRGreenⅠKey words
peste des petits ruminants virus/conventional RT-PCR/Real-time quantitative RT-PCR/SYBR Green Ⅰ分类
农业科技引用本文复制引用
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):2844-2851,8.基金项目
国家自然科学基金项目(31172342) （31172342）
国家科技支撑计划(2013BAD12B05) （2013BAD12B05）
转基因重大专项(2014ZX0801203B) （2014ZX0801203B）
