生物素标记寡核苷酸探针原位检测石蜡切片鸭瘟病毒核酸  

Studies on the in situ detection of duck plague virus nucleic acid in paraffin section by biotin labelled oligo nucleotide probe

作　　者：程安春[1,2] 廖永洪[1] 汪铭书[1,2] 袁桂萍[1,2] 徐超[1,2] 韩晓英[1] 郭宇飞[1,2] 

机构地区：[1]四川农业大学动物科技学院禽病防治研究中心,四川雅安625014 [2]四川省动物疫病与人类健康重点实验室,四川雅安625014

出　　处：《中国兽医学报》2009年第4期403-408,共6页Chinese Journal of Veterinary Science

基　　金：教育部"新世纪优秀人才支持计划基金资助项目"(NCET-04-0906);四川省"十五"攻关重大生物技术基金资助项目(01NG018-01);四川省重点建设学科基金资助项目(SZD0418)

摘　　要：据GenBank中有关鸭瘟病毒(DPV)的1个765 bp的EcoRⅠ片段序列,用Oligo软件设计长度为37 bp的寡核苷酸并用生物素标记制备探针,经blast分析和斑点杂交检测探针的特异性后,建立从石蜡切片中检测出DPV核酸的原位杂交方法并对人工感染死亡鸭的各组织器官进行检测,结果显示:(1)寡核苷酸探针能特异性检测到DPV强毒CHv株DNA,对鸭病毒性肝炎病毒QL79株RNA、血清1型鸭疫里默氏杆菌DNA、鸭源多杀性巴氏杆菌DNA、鸭沙门菌DNA和大肠杆菌DNA的检测结果为阴性。(2)以寡核苷酸探针建立的从石蜡切片中检测出DPV核酸的原位杂交方法的最佳反应条件为:组织切片先用0.2 mol/L HCl 37℃处理20 min,然后用100 mg/L的蛋白酶K 37℃消化15 min左右;杂交时探针工作浓度为350μg/L;Avidin-AP的工作稀释度为1∶100。(3)以所建立的原位杂交法检测DPV-CHv强毒人工感染死亡鸭的各组织器官,结果肝脏、肠道、法氏囊、脾脏、食道、肺脏和肾脏呈阳性反应,DPV的DNA分布于特定细胞的细胞浆和细胞核。结果表明,原位杂交检测石蜡切片中DPV的方法具有直观、特异性强的优点,是对DPV进行检测和病原定位的良好方法,可用于DPV的侵染过程和致病机理研究及回顾性诊断检测。To establish methods that could be employed to study the position of duck plague virus nucleic acid in paraffin section so that effective method could be used in duck plague virus infectivity and pathogenesis investigation and looking back diagnosis. Duck plague virus nucleic acid in situ detection method was established after the specificity of a biotin labelled 37 bp oligo nucleotide probe which was designed by the software of Oligo according to a 765 bp EeoR I digested DPV DNA fragment sequence in GenBank was verified by blast analysis and dot blotting,and by this method specimen of tissues and organs from ducks which underwent mortality were examined. The oligo nucleo-tide probe could specifically detect DNA of DPV-CHv strain but the results of detection on RNA of duck virus hepatitis virus QL79 strain,DNA of duck Riemerella anatipestifer serotype 1, DNA of duck Pasteurella multocida,DNA of Salmonella anatum,DNA of E. coli were all negative. The optimum detection protocol was as follows： sections of tissues were firstly treated by 0. 2 mol/L HCI at 37°C and then digested by 100 mg/L protease K at 37°C for 15 min;At hybridization the probe working concentration was 850 /μg/L and the Avidin-AP dilution factor of was 1 ： 100. The tissues and organs of ducks showing morbidity in artificial infection were examined by this established method and the results indicated that the specimens of liver, intestine, bursa Fabricius, spleen, esophagus, lung and kidney were positive and the DPV DNA distributed in cytoplasm and nucleus of specific cells. The method of in situ hybridization to detect DPV in paraffin section was specific and easy to judge and was excellent in detecting DPV and investigating lhe position of DPV in tissues. This method could be used in DPV infectiveness ＆ pathogenesis investigation and looking back diagnosis.

关 键 词：鸭瘟强毒 原位杂交 检测 分布 

分 类 号：S852.65[农业科学—基础兽医学]