【作者】 刘苹； 杨金堂； 王红宁；

【Author】 LIU Ping, YANG Jin - tang, WANG Hong - ning (Animal Science Department, School of Agriculture , Shanghai JiaoTong University, Shanghai 201101, China; Ningbo Chia - Tai Agriculture LTD. Co Zhe jing Ningbo 315806, China; Shi chuan Agriculture College, shichuan Yaan 612504,China)

【机构】 上海交通大学农学院； 宁波正大农业有限公司； 四川农业大学；

【摘要】 提取IBV标准强毒株M41 S1基因的重组质粒PIBV S1 M41,经HindⅢ、BamHI双酶切后电泳,低熔点琼脂糖回收IBV S1基因cDNA片段,经紫外分光光度计计算其含量为 100ng／μl,用地高辛素标记物(DIG-11-dUTP)对回收的IVB S1全基因(1．72kb)进行标记。其标记含量经测定为10ng/μl,将标记的DIG IBV S1基因探针与已知IBV的12株病毒 RNA核酸提取物进行斑点杂交反应,其中包括4株标准株H52、H120、M41、T株和8株分离株,其结果该探针与12株已知。IBV毒株均成阳性反应。交叉试验:该探针与NDV、IBDV、 ILTV、正常胚液、蒸馏水对照均成阴性,说明该探针具有较强的群特异性。该探针的检测灵敏度为9．77fg,杂交灵敏度为24．41pg,具有很高的敏感性。作者建立的IBV检测方法与传统的 IBV检测方法相比,具有方便、快速、敏感、特异的优点,是实验室确诊IBV的一种先进方法。更多还原

【Abstract】 In this study, recombinant plasmid PIBV S1 M41 including IBV S1 Gene was extracted , The plasmid was then digested by Hind Ⅲ and BamHI restriction enzyme and then separated by gel electrophoresis. IBV S1 cDNA was recovered and concentration was 100ng/μl then the total IBV S1 Gene (1.72kb) was labeled by the randomly primed incorporation of digoxigenin - dUTP and the concentration of the DIG - labeled probe DNA was 10ng/μl. The identity of the products was verified by hybridization to tested productsand specfic DIG - labeled DNA probe. Twelve of tested produts including four standard strains (H120、H52、M41、T) and eight isolation strains (SHIB、XJIB、SAIB1、SAB2、SAIB3、SAIB4、SAIB5、D41) obtained by ampliftion of RNA extracted from the allantioc fluid were positive. But no hybridization signal was exhibited in other spectments such as NDV、IBDV、ILTV、normalallantioic fluid and ddH2O. The senitivity of probe dectection and Hybrizdzation. were 9.77fg and 24.41pg. It was strongly indicateed in this experiment that the DIG-labeled IBV S1 cDNA probe to be convenient, fast, high labelled rate and highly spectfic method of detection.更多还原