应用DIG标记探针杂交检测IBDV

Establishment of Hybridization Assay With Nucleic Acid Probe to Detect IBDY and Comparison with Immunodiffusion Test

本试验用地高辛（Ｄｉｇｏｘｉｇｅｎｉｎ，ＤＩＧ）标记的探针建立了核酸杂交检测传染性法氏囊病病毒（ＩＢ－ＤＶ）的方法，并在敏感性方面同琼脂扩散试验进行了比较。探针来源于ＩＢＤＶＳＴＣ－ｌｌ９和ＳＴＣ－２４３ｃＤＮＡ。杂交方法的敏感性试验表明，核酸杂交可以检测到０．１ｐｇ的ＩＢＤＶＲＮＡ；与多个毒株核酸的杂交则显示出标记的探针可以作为通用试剂用于ＩＢＤＶ早期感染的诊断；而同琼脂扩散试验的比较则说明，杂交方法比常规的免疫沉淀反应敏感１０４倍以上。除此之外，由于杂交方法特异以及非放射性探针操作方便，因而具有很大的应用前景。

he hybridization assay with digoxigenin(DIG)-labeled probe was developed for detection of in-fectious bursal disease virus(IBDV)and it's semitivity was compared with that of immunodiffusiontest in this experiment.IBDV STC-119 and STC-24 3 cDNA fragments used as probe were preparedfrom recombinant pUC plasmid with Hind Ⅲ and EcoRI and labeled with DIG.IBDV RNA was ex-tracted from IBDV purified from chicken bursa which were homogenized followed by extraction withchloroform,precipitation of virus with immune serum,digestion with proteinase K and recovery of IBDV RNA with ethanol precipitation.The results show that 0.lpg of IBDV RNA can be positively de-tected by this method and the mixed probe were able to hybridize with RNA from several IBDV isolatesand can be used for diagnosis of infectious bursal disease in the early stage of infection.The comparisonwith immunodiffusion test revealed that the furmer is l04 times more sensitive than the later,More-over,the specificity and the convenience of nonradioactive probe make the hybridization assay a veryuseful method in veterinary diagnostic laboratory.