多杀性巴氏杆菌TaqMan荧光定量PCR检测方法的建立与应用  

Development and application of Taq Man real-time PCR assay for the detection of Pasteurella multocida

作　　者：许腾林 邢桂玲 刘家森[1] 刘大飞[1] 李志杰[1] 姜骞[1] 康洪涛 田进[1] 郭东春[1] 曲连东[1] XU Teng-lin;XING Gui-ling;LIU Jia-sen;LIU Da-fei;LI Zhi-jie;JIANG Qian;KANG Hong-tao;TIAN Jin;GUO Dong-chun;QU Lian-dong(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,China;State Key Laboratory of Veterinary Bintechnology,Harbin Veterinary Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;College of Animal Science and Technology,Northeast Agricultural University,Harbin 150030,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 [2]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [3]东北农业大学动物科技学院,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2018年第8期706-710,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2016YFD0500800)

摘　　要：为建立一种多杀性巴氏杆菌(Pm)快速敏感的检测方法,本实验根据Pm70株kmt1基因保守区设计特异性引物和TaqMan探针,建立了该菌的TaqMan荧光定量PCR检测方法,并优化了检测反应条件。本实验建立的荧光定量PCR方法可以特异性检测Pm,而对鸭疫里默氏杆菌、支气管败血波氏杆菌等菌株检测结果均为阴性,表明该方法具有良好的特异性。该方法对C48-1株标准品的检测灵敏度为1.75×10~1拷贝/μL,优于常规PCR检测方法(1.75×10~3拷贝/μL) 100倍;组内和组间重复性试验的变异系数均小于2%,具有良好的重复性。对20份疑似感染的临床病料样品(鸡肝脏、牛肺脏)进行增菌培养和分离鉴定,结果分离到8株多杀性巴氏杆菌;以建立的荧光定量PCR方法与常规PCR方法对增菌培养液进行检测,阳性率分别为40%(8/20)和25%(5/20),且荧光定量PCR方法与常规细菌分离鉴定的符合率为100%。对C48-1株感染致死的20份小鼠肝脏和脾脏组织研磨液进行检测,阳性率均为100%。本研究建立的方法能够快速、敏感的检测Pm,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 method for Pasteurella multocida detection, the Taq Man real-time PCR was established with a pair of primers and probe targeting the conserved region of kmt1 gene of P.multocida Pm70 strain. The assay was highly specific for amplification from P.multocida, but no amplification from Riemerella anatipestifer, Brodetella bronchiseptica and so on. The detection limitation of the protocol was 1.75 ×10 copies/μL initial template, which was 100-fold more sensitive than that of conventional PCR. Moreover, the method was highly reproducible and a coefficient of variation was less than 2% for both intra-and inter-assay. Tweenty tissue samples collected from chicken（livers） and bovine（lungs） were detected, and 8 samples were positive, while the positive rate of detection was 45%（8/20） by the real-time PCR and 25%（5/20）by conventional PCR, respectively. Positive rates of 20 samples from livers and spleens of mice infected with P.multocida C48-1 strain were 100% by both real-time PCR and conventional PCR. Therefore, the real-time PCR assay provided a sensitive and rapid method for detection of P.multocida in the laboratory and clinical infection.

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]