动物医学进展2016，Vol.37Issue(4)：1-5,5.
SYBR Green Ⅰ实时荧光定量 PCR 检测 PRRSV 方法的建立
Establishment of SYBR Green Ⅰ Real-time Fluorescence Quantitative PCR Method for Detection of PRRSV
摘要
Abstract
Primers were designed for SYBR Green Ⅰ real-time fluorescence quantitative PCR according to the PRRSV M protein.Different temperature gradient results were checked to optimize the reaction sys-tem.The specificity and stability of the detection method were verified through different genera of common swine pathogenic RNA virus vaccine and 2 representative genetype vaccine strains which were selected on the basis of the domestic molecular epidemiological characteristics of PRRSV.A standard curve was con-structed and the sensitivity of this detection method was explored with the plasmid which was diluted in 10 times of gradient.10 conserved PRRSV positive samples and 10 conserved PRRSV negative samples were selected randomly to research the coincidence rate of the constructed detection and quarantine standards recommended by the Ministry of agriculture.The results showed that detection could be completed within 80 min when the temperature was set at 54.4℃,using the two-step detection system.The detection method has good specificity and stability.The detection sensitivity could as low as 6.8×102 copies/L.The standard curve was the equation (y=-3.411x+39.002,R2 = 0.999)when the Ct value was selected as the abscis-sa and the amplified sequences of numerical was selected as ordinate.The detection result was in line with the quarantine standards recommended by the Ministry of Agriculture with the randomly selected porcine samples.The test results showed that the SYBR Green Ⅰ real-time fluorescence quantitative PCR method was established successfully which could provide a good means for clinical detection.关键词
猪繁殖与呼吸综合征病毒/M 蛋白/SYBR Green Ⅰ实时荧光定量 PCRKey words
PRRSV/M protein/Green Ⅰ SYBR real-time fluorescence quantitative PCR分类
农业科技引用本文复制引用
罗忠永,王印,杨泽晓..SYBR Green Ⅰ实时荧光定量 PCR 检测 PRRSV 方法的建立[J].动物医学进展,2016,37(4):1-5,5.基金项目
“十二五”国家科技支持计划项目 （）
