摘要
本试验研制了光敏生物素标记鸭瘟病毒全基因组核酸探针,对此探针的敏感性和特异性进行了研究。把提纯的鸭瘟病毒经蛋白酶消化后,提取核酸,核酸和光敏生物素经光标记后制成核酸探针。经亲和素—辣根过氧化物酶(AV-HRP)或碱性磷酸酶显色的核酸斑点杂交结果表明:该探针最低可检出10 pg(AV—AP法)同源的鸭瘟病毒F_(33)—DNA或10^(-5)稀释的攻毒鸭脑中的F_(33)—DNA;用辣根酶系统显色,灵敏度要比碱性磷酸酶的低约10倍;该探针与鸭瘟弱毒、禽巴氏杆菌、鸭肝炎病毒、新城疫病毒、马立克氏病毒和正常鸭组织均不出现杂交信号,表明探针是敏感与特异的;探针在—18℃的保存期大于4个月;用探针检测21份病料,阳性率为90.5%(19/21),其中攻毒病料100%(7/7)被检出,而病毒分离—电镜法的检出率仅为75%(6/9)。杂交结果还显示了病鸭体内各组织含毒量的大小,依次是:脑>肝,脾>血,肾>肌肉。
In this experiment, a duck plague virus (DPV) DNA probe labelled by photobiotin has been developed, and the sensitivity and specificity of the probe were studied. DPV-DNA was isolated from purified virus particles which has been digested by pronase. The probe was prepared by labelling DPV-DNA with photobiotin under a special light source. The DNA dot hybridization tests whose reactions were carried out by AV-AP dying systems show that the probe could detect homologous DNA at 10 pg purified DNA level or at 10^(-5) fold dilution of duck plague's duck brains treated with pronase. The sensitivity of AV-HRP system was 10 times lower than that of AV-AP system's. No hybridization signal was found in other specimens such as avirulent strain of DPV, duck hepatitis virus, NDV, MDV or healthy duck tissue. Thus, the probe was sensitive and specific in diagnosis. It also showed that the probe could be safely stored at-18℃ for at least 4 monthes. 21 specimens were detected by means of the probe, and the positive rate meet 90.5% (19/20). Out of them, 7 positive signals were 100% found in 7 specimens of duck tissue inoculated by homologous strain of DPV. But only 75% (6/9) of the specimens was detected positively by isolation-eletron microscopy. The hybridization test also showed that different kinds of duck tissues had different quantity of DPV particles: brains>liver, spleen>blood, kidney>muscle.
关键词
鸭病
鸭瘟病毒
光敏生物素
photobiotin
nucleic acid probe
hybridization
duck plague virus
detection
