检测石蜡切片中鸭病毒性肠炎病毒间接原位PCR方法的建立  

Development and Application of Indirect in situ Polymerase Chain Reaction for Detecting Duck Enteritis Virus in Paraffin Sections

作　　者：程安春[1,2] 廖永洪[1] 朱德康[1,2] 汪铭书[1,2] 罗启慧[1,2] 贾仁勇[1,2] 陈孝跃[1,2] 

机构地区：[1]四川农业大学动物医学院禽病防治研究中心,四川雅安625014 [2]动物疫病与人类健康四川省重点实验室,四川雅安625014

出　　处：《中国农业科学》2008年第12期4365-4371,共7页Scientia Agricultura Sinica

基　　金：教育部"新世纪优秀人才支持计划"项目(NCET-04-0906/NCET-06-0818);国家自然科学基金项目(30771598);高等学校科技创新工程重大项目培育资金项目(706050);国家"十一五"科技支撑计划(2007Z06-017);四川省十五攻关重大生物技术项目(01NG018-01);四川重点攻关项目(05NG002-003);四川省基础研究项目(07JY029-016/07JY029-017)

摘　　要：【目的】建立能对石蜡切片中鸭病毒性肠炎病毒(DEV)核酸进行定位的原位PCR方法,为鸭病毒性肠炎(DVE)存档蜡块的回顾性诊断、致病机理研究等提供有效的实验手段。【方法】据DEV的UL30-UL31基因序列设计PCR引物和寡核苷酸探针,以DEV感染死亡鸭肝脏组织石蜡标本制作切片,经蛋白酶K消化、原位PCR扩增和生物素标记的寡核苷酸探针原位杂交,建立了检测石蜡标本中DEV的间接原位PCR方法并应用于人工感染DEV不同时间的鸭肝脏、DVE发病鸭的存档蜡块和临床病料检测。【结果】间接原位PCR对DVE死亡鸭肝脏的石蜡标本检测结果为阳性,而鸭病毒性肝炎、鸭疫里默氏杆菌病、鸭多杀性巴氏杆菌病、鸭沙门氏菌病和鸭大肠杆菌病死亡鸭肝脏的石蜡标本检测结果为阴性;间接原位PCR对人工感染DEV后2、4、6、12、24、48和72 h不同时间的鸭肝脏检测结果均为阳性,阳性细胞有肝细胞、窦皮细胞和枯否氏细胞,阳性信号多出现于坏死细胞的碎片中或细胞坏死后形成的空泡内及空泡边缘;对存档蜡块、临床病料的检测与病毒分离鉴定吻合率为100%。【结论】本研究建立的间接原位PCR方法具有直观、敏感、特异性强的优点,在显示核酸阳性信号的同时,还能判别含有靶序列的细胞类型以及组织细胞的形态结构特征与病理变化。可用于DVE的诊断、分子流行病学调查、存档蜡块的回顾性诊断和致病机理的研究。[Objective] To establish methods that could be used to study the position of duck enteritis virus nucleic acid in paraffin section so that effective method could be used in duck enteritis virus infectivity and pathogenesis investigation and retrospective diagnosis. [ Method ] A pair of primers and an oligonucleotide probe were designed by Oligo software according to the UL30-UL31 sequence of duck enteritis virus in GenBank. The specificity of the primers was tested by PCR. The specificity of probe was tested by dot-hybridization. Indirect in situ polymerase chain reaction （IS-PCR） was developed for detecting duck enteritis virus in paraffin sections after digesting with protease K, DNA in situ amplification and in situ hybridization （ISH）. [Results] The indirect IS-PCR main conditions were optimized as follows： tissue sections were treated by 0.2 mol/L HCI at 37℃ for 20 minutes, by 100 μg·ml^-1 protease K at 37℃ for 15 minutes; The working condition of probe and Avidin-AP of ISH were 350 ng·ml^-1 and 1 ： 100, respectively. The positive results were gained by indirect IS-PCR for detecting the duck liver paraffin sections infected by DEV, but negative results to the duck liver paraffin sections of dead ducks infected with duck viral hepatitis virus, Riemerella anatipestifer,Pasteurella multocida, Salmonella typhimurium, E.coli （O78）. The positive signals were detected from hepatocyte, sinusoidal endothelial cell and Kupffer＇s cell of duck liver infected artificially with DEV by indirect IS-PCR at 2, 4, 6, 8, 12, 24, 48 and 72 h, respectively. Most of them appeared in debris of cellular necrosis or vacuole formed in infected cells. Five archival paraffin and eighteen clinical samples which came from ducks infected with DEV were detected, the results of indirect IS-PCR and isolate identification method were identical completely. [Conclusion] The results show that the method is rapid, sensitive and specific for detecting DEV. The method could be widely used for diagnosis, molecular epidemiolog

关 键 词：鸭病毒性肠炎病毒 原位PCR 寡核苷酸探针 石蜡标本 检测 

分 类 号：S852.65[农业科学—基础兽医学] TS207.3[农业科学—兽医学]