鸭疫里默氏杆菌荧光定量PCR检测方法的建立  

Establishment of real-time PCR for detection of Riemerella anatipestifer

作　　者：冯金牛[1,2] 陈芳艳[3] 晏永邦[2] 张秀[2] 黎丁滔[2] 区德庆[1] 唐秀英[1] 陈瑞爱[1] 王林川[1,2] 

机构地区：[1]广东大华农动物保健品股份有限公司,广东新兴527400 [2]华南农业大学兽医学院,广东广州510642 [3]华南农业大学动物科学学院,广东广州510642

出　　处：《中国兽医科学》2011年第4期392-396,共5页Chinese Veterinary Science

基　　金：广东省科技攻关项目(2006A20301001)

摘　　要：根据鸭疫里默氏杆菌(RA)16SrRNA基因的保守序列设计特异性引物和TaqMan探针,建立了检测RA的实时荧光定量PCR方法。特异性试验结果显示,2株RA均呈阳性,而3株非RA均呈阴性。试验结果表明,该方法具有良好的特异性,该方法的表达式Ct=-3.676×lg copies+45.17,相关系数r2=0.996,R循环效率Eff(%)=93.482,DNA拷贝数在100～108范围内检测曲线有良好的线性关系,对质粒标准品的最低检测限为1.0×101 copies/μL,重复性检测的变异系数低于5%,利用该方法分别对自然感染表现典型鸭疫里默氏杆菌病临床症状和病理变化的死亡雏鸭肝、心、脑组织等不同脏器进行细菌定量检测,阳性率均为100%,而用普通PCR检测阳性率分别只有72%(18/25)、84%(21/25)和92%(23/25)。证实本研究所建立的荧光定量PCR方法具有敏感性高、特异性好、稳定性强的特点,可用于RA感染的快速诊断、流行病学调查以及鸭产品的检疫。A pair of primers and one TaqMan probe were designed according to the conserved region of the 16 S rRNA gene of Riemerella anatipestifer（RA）,and a rapid fluorescent quantitative PCR（FQ-PCR） assay was successfully established for detection of RA.In result,specificity test showed that 2 RA strains produced positive reactions,while 3 other non-RA bacterial strains produced negative reactions.The equation of the assay was Ct=-3.676 lg copies ＋ 45.17,and the correlation coefficient was 0.996 and PCR efficiency of crying reached up to 93.482%.There was an excellent linear correlation during the DNA concentration from 1 copy to 1×108 copies.The sensitivity of the assay was 1.0×101 copies/μL,and the vari-ation coefficient was less than 5%.Comparison of RA conventional PCR with FQ-PCR for detection of the samples of the heart,liver and brain from dead duckling infected with RA,the positive rates by RA conventional PCR were 72%,84% and 92% respectively,but 100% positive for all samples by FQ-PCR.Thus,this FQ-PCR assay had high sensitivity,specificity and stability,which could be used for the rapid detection,epidemiological investigation and duck product quarantine of RA infection.

关 键 词：鸭疫里默氏杆菌 荧光定量聚合酶链反应 TAQMAN探针 

分 类 号：S852.612[农业科学—基础兽医学]