副结核分枝杆菌DNA探针的制备与应用  

Preparation and Using of the DNA Probe of Mycobacteri-um Paratuberculosis

作　　者：刘思国[1] 刘清何 陈奖励[1] 李卯年[1] 何昭阳[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所,吉林农业大学动物科学系

出　　处：《中国兽医学报》1996年第1期43-47,共5页Chinese Journal of Veterinary Science

基　　金：吉林省科委资助项目

摘　　要：以溶菌酶、ＳＤＳ、高氯酸钠等处埋提取副结核分枝杆菌Ｃ２株染色体ＤＮＡ，经限制性内切酶ＰｓｔⅠ消化后，以质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ为载体，通过Ｔ４ＤＮＡ连接酶连接，转入Ｅ。ｃｏｌｉＤＨ５ａ受体菌中，构建了副结核菌Ｃ２株的ＤＮＡ基因文库。应用反向杂交试验，从基因文库中筛选出４个重组克隆：ＰＴＰ１２、ＰＴＰ１９、ＰＴＰ３１、ＰＴＰ３８。对这４个重组克隆进行酶切、电泳分析，结果表明４个插入片段长度分别为：４．０ｋｂ、１．８ｋｂ、１．３ｋｂ、２．３ｋｂ。用光敏生物素将此４个插入片段标记成ＤＮＡ探针。通过斑点杂交试验，从４个探针中筛选出１个特异性探针ＰＴＰ３１。该探针与副结核分枝杆菌呈强杂交反应，除与堪萨斯分枝杆菌、胞内分枝杆菌Ⅲ型呈弱杂交反应外，与其他受试的分枝杆菌及大肠杆菌等非分枝杆菌均不反应。ＰＴＰ３１探针的敏感性为２ｎｇ的ＤＮＡ和１０５个细菌。以ＤＮＡ探针、粪检菌、ＥＬＩＳＡ３种方法分别对３２份副结核菌素（ＰＰＤ）变态反应阳性牛粪便及血清样品进行检测，其检出率分别为４７％（１５／３２）、５６％（１８／３２）和３４％（１１／３２）。该探针与粪检菌的符合率为６７％（１２／１８）。对随机采取的２７６份牛粪便及血清?A genomic library of chromosomal DNA extracted from one cattle strain(C2)of Mycobacterinm paratuberculosis was constructed in pBluescript SK vector.Thelibrary was screened by reversed plague hybridization with labelled Mycobacteriumparatuberculosis genomic DNA as probe,and four DNA probes (designated as PTP12,PTP19,PTP31,PTP38 respectively)were obtained.One clone of the four probes specificfor Mycobacterium paratuberculosis was detected by using Dot-hybridization,DNA fromthis clone could detect 2ng Mycobacterium paratuberculosis DNA and 105 bacteria in fe-ces.The fecal and serum samples collected from 32 paratuberculosis-infected cattle thosewere detected with Johnin test were examined simultomeausly by using the probe,mi-croscopical examination and ELISA.The positive rates were 47%(15/32), 56%(18/32)and 34%(11/32)respectively, and the positive rates of fecal and serum samplescollected randomly from 276 clinically healthy cattle(Johnin test negative)were detect-ed by those diagnostic methods were 10%(27/276), 13%(36/276)and 7%(19/276)respectively.The conformity rate of the diagnostic results obtained by the probe and mi-croscopical examination was 67%(12/18).

关 键 词：核酸探针 副结核菌 ELISA 病原细菌 

分 类 号：S852.61[农业科学—基础兽医学]