同时检测多杀性巴氏杆菌及其荚膜A型的双重TaqMan荧光定量PCR检测方法的建立与初步应用  

Development and application of duplex TaqMan real-time PCR assay for the detection of Pasteurella multocida and its capsular type A

作　　者：刘本金 郑亚婷 许腾林 姜骞[1] 刘家森[1] 康洪涛 田进[1] 郭东春[1] 曲连东[1] LIU Ben-jin;ZHENG Ya-ting;XU Teng-lin;JIANG Qian;LIU Jia-sen;KANG Hong-tao;TIAN Jin;GUO Dong-chun;QU Lian-dong(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2021年第5期501-506,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2016YFD0500800)。

摘　　要：为建立可以同时检测多杀性巴氏杆菌(Pm)及其荚膜A型的快速敏感检测方法,本研究在建立的Pm kmt1基因的荧光定量PCR基础上,设计了该菌capA基因特异性引物和Taq Man探针,经优化反应条件建立了双重Taq Man荧光定量PCR检测方法。本实验从荚膜A型Pm C48-1菌株中扩增了capA基因,构建了重组质粒pMDcapA;以pMD-capA重组质粒为标准品,建立的标准曲线在9.83×10^(3)拷贝/μL~9.83×10^(9)拷贝/μL内与Ct值具有良好的线性关系,相关系数(R^(2))为0.9873,扩增效率(E)为1.032。建立的双重荧光定量PCR方法可以同时检测Pm及其荚膜A型,而对B型、D型Pm、鸭疫里默氏杆菌、支气管败血波氏杆菌等细菌检测结果均为阴性,具有较强的特异性;对重组质粒标准品的检测灵敏度为10^(1)拷贝/μL,高于常规PCR检测方法(10^(3)拷贝/μL)100倍具有较高的敏感性;组内和组间重复性试验的变异系数均小于2%,具有较好的重复性。本实验建立的双重荧光定量PCR方法可以同时快速敏感地检测Pm及其荚膜A型,对于其临床和实验室的快速诊断具有重要意义。To develop a rapid and sensitive detection method of Pasteurella multocida and its capsular type A simultaneously,a duplex real-time quantitative PCR method was developed and optimized with primers and probe basing on the capA gene.The capA gene was amplified using P.multocida C48-1 strain genomic DNA(gDNA)as template and the recombinant plasmid pMD-capA was constructed as standard plasmid.The standard curve was linear in the range of 9.83×10^(3)copies/μL to 9.83×10^(9)copies/μL,and the correlation coefficient(R2)was 0.9873,and the amplification efficiency(E)was 1.032.The established duplex TaqMan real-time PCR could simultaneously detect Pasteurella multocida and its capsular type A,but it is negative for Pasteurella multocida capsular type B and D,Riemerella anatipestifer,Bordetella bronchiseptica and other bacteria,indicating high specificity.The sensitivity test demonstrated that the detection limit of protocol for standard plasmid pMD-capA was 10^(1) copies/μL,which was 100-fold more sensitive than that of conventional PCR(10^(3)copies/μL),demonstrating a high sensitivity.Moreover,this method was highly reproducible and a coefficient of variation was less than 2%for both intra-assay and inter-assays.Thus,the duplex TaqMan real-time PCR developed in this study provides a sensitive and rapid method for detection of P.multocida and its capsular type A simultaneously,which lays a foundation for the rapid diagnosis in the laboratory and clinical settings.

关 键 词：多杀性巴氏杆菌 TAQMAN探针 荧光定量PCR 

分 类 号：S852.61[农业科学—基础兽医学]