马立克氏病两种诊断方法的建立和应用

DEVELOPMENT AND APPLICATION OF TWO DIAGNOSTIC TECHNIQUES ON MAREK'S DISEASE

本试验建立了诊断马立克氏病(MD)的两种方法——斑点酶联免疫吸附试验(Dot-ELISA)和斑点杂交法(DOt-blot hybridization,DB),并与琼脂扩散试验(AGP)进行了比较。用AGP和Dot-ELISA对122份羽毛样品进行检测,MD阳性率分别为35.3％和54.9％,经统计学分析,两者差异极显著。AGP、Dot-ELISA和DB对其中27份样品的检出率依次为29.6％,40.7％和44.4％,三者的相关系数为0.84。生物素标记的MDV基因的G、H、N三个片段与各个MDV毒株杂交的结果是类似的。生物素探针的灵敏度可达pg水平,保存6个月的探针英活性不受影响。探针与宿主核酸之间存在微弱杂交,可与质粒栽体杂交。

Dot-enzyme-linked immuno-sorbent assay (Dot-ELISA) and Dot-hybri dization were developed and applied to detect the antigen and DNA of Marek's disease virus (MDV).A5.5-kilobase BamHI fragment of MDV gcne library was used as a probe. Extensive homology existed between MDV and hcrpesvirus of the turkey (HVT) when biotin-labeled probe was used for Dot-blot hybridization. Specific hybridization between diotin-labeled pBR322 DNA and DNA from various clinical samples was found. Of 19 samples from chickcns infected with MDV, the detection percentages using AGP, Dot-ELISA and Dot-blot hybridization were 78.9% , 84.2% and 89.5% respectively. Of 122 clinical samples to be tested, 53 were negative and 41 were positive by both AGP and Dot-ELISA, 26 samples were Dot-ELISA positive but AGP negative. Of 27 samples, 14 were negative and 8 were positive by three methods. There is a good correlation among different tests.