基于鸽新城疫病毒M基因实时定量PCR检测方法的建立  

Establishment of real-time quantitative PCR detection method based on M gene of Pigeon Newcastle disease virus

作　　者：刘承惠 郑百利 徐静怡 周铁忠 李冰 Liu Chenghui;Zheng Baili;Xu Jingyi;Zhou Tiezhong;Li Bing(College of animal husbandry and veterinary medicine,Jinzhou Medical University,Liaoning Jinzhou 121001;Muyuan Food Co.,Ltd,Henan Nanyang 473000)

机构地区：[1]锦州医科大学畜牧兽医学院,辽宁锦州121001 [2]牧原食品股份有限公司,河南南阳473000

出　　处：《现代畜牧兽医》2020年第4期14-19,共6页Modern Journal of Animal Husbandry and Veterinary Medicine

基　　金：辽宁省自然科学基金计划指导项目(2019-ZD-0827)。

摘　　要：研究通过比对鸽源和鸡源新城疫病毒M基因的序列,找到其差异位点并在保守区设计引物和TaqMan探针,建立了一种可以特异性检测鸽新城疫病毒的实时荧光定量PCR方法。以鸽新城疫病毒M基因阳性质粒为模板制作标准曲线并对检测方法的指标进行系统评价。结果显示,该方法的标准曲线为方程为:y=-3.334logX+37.837,相关系数R^2=0.992;重组质粒标准品检测下限为1×10^1 copies/μL,比普通PCR灵敏100倍;特异性试验显示,该方法与鸡新城疫病毒不同毒株及其他常见禽类病毒无交叉反应;重复性试验的组间及组内的变异系数均小于2%,说明本研究建立的实时定量PCR检测方法敏感性高、特异性强、重复性好,可应用于鸽新城疫病毒的早期检测,为鸽新城疫病毒的防控提供了技术支持。In this study,by comparing the sequences of Newcastle disease virus M genes from pigeon and chicken sources,finding their differential sites,and designing primers and Taq Man probes in the conserved region,we established a real-time fluorescent quantitative PCR method to detect Newcastle disease virus in pigeon.The standard curve was made with the positive plasmid of pigeon Newcastle disease virus M gene as template and the indicators of this detection method were evaluated systematically.The results showed that the standard curve of the method was the equation:y=-3.334 log X+37.837,the correlation coefficient R^2=0.992,the detection lower limit of the recombinant plasmid standard was 1×10^1 copies/μL,which was 100 times more sensitive than ordinary PCR sensitizers;Specific tests showed that this method had no cross-reaction with different strains of chicken new epidemic virus and other common avian viruses;the coefficient of variation between groups and within groups was less than 2%,which indicates that the real-time quantitative PCR detection method established in this study has high sensitivity,strong specificity and good repeatability,and it thus can be used in the early detection of Newcastle disease virus in pigeons,providing technical support for the prevention and control of Newcastle disease virus in pigeons.

关 键 词：新城疫病毒 M基因 实时荧光定量PCR 方法建立 鸽 

分 类 号：S854.4[农业科学—临床兽医学]