【作者】 宋涌；

【导师】 程安春； 汪铭书；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2003， 硕士

【摘要】 本文根据基因库（Gene Bank）登载的鸭瘟病毒（Duck Plague Virus，DPV & Duck Enteritis Virus，DEV）基因组中的一个765bp长的EcoRⅠ片段（Anatid Herpesvirus 1 DNA Polymerase Gene & Unknown Gene），运用软件OLIGO设计一对引物，其扩增幅度为498bp。应用这对引物针对鸭瘟病毒（DPV）DNA进行扩增，获得了与设计大小相符合的特异性条带，成功建立了检测鸭瘟病毒的PCR方法。确立了PCR的最佳反应条件，引物浓度为0.5μM，Mg²⁺作用浓度为2.5μM；最佳反应模式为：95℃变性5min，进入94℃1min，51.8℃1min，72℃2min的循环，共进行35个循环，最后经72℃延伸10min后于4℃结束反应。特异性试验表明，对七种对照禽病病原体（鸡马立克氏病病毒、鸡传染性喉气管炎病毒、鸭病毒性肝炎病毒、鸭巴氏杆菌、鸭大肠杆菌、鸭副伤寒沙门氏菌、鸭疫里默氏杆菌）的基因却扩增不出任何条带，为阴性。敏感性试验表明，可检测到10fg的鸭瘟病毒DNA模板。 本文研究了鸭瘟病毒弱毒Cha株经皮下、口服和滴鼻三种途径接种含母源抗体的雏鸭后，应用本文建立的PCR方法检测不同时间鸭瘟弱毒在鸭体各组织器官的分布规律。分别在接种后的第4h、8h、12h、24h、3d、5d、7d、10d、14d、17d和21d人工致死雏鸭，收集雏鸭的静脉血液、心、肝、脾、肺、肾、十二指肠、直肠、法氏囊、胸腺、胰腺、延脑、大脑、小脑、舌头、肌肉、骨髓、粪便和食道共19种样品进行PCR检测。试验结果表明：①皮下组在接种后的第4h，即可在心、肝、脾、肾、法氏囊、胸腺、胰腺、延脑、大脑和小脑共10种组织中检出DPV的DNA，8h后所有采取的组织器官均可检测到DPV的DNA；②口服组最早在接种后4h可在舌头和食道中检测到DPV的DNA，8h后可在心、肝、脾、肾、胸腺、胰腺、延脑、大脑、小脑、舌头、食道和血液共12组织器官中检出DPV的DNA；③滴鼻组在接种后4h未能在组织中检出DPV的DNA，8h后可在心、肝、脾、肾、胸腺、延脑、大脑、小脑、舌头、食道和血液共11中组织中检测到DPV的DNA。④在三种途径中，检出时间最早和检出率最高的组织器官为肝脏、脑（大脑、小脑和延脑），至第21天时均能稳定、持续地从所采取的组织中检测出DPV基因组。 本研究建立的PCR方法适合于作为鸭瘟病毒感染的实验室诊断；应用本研究建立的PCR诊断方法检测鸭瘟病毒弱毒株在雏鸭体内的分布规律，从而为临床上更好地应用鸭瘟病毒弱毒株预防鸭瘟提供了有力的理论依据。更多还原

【Abstract】 A polymerase chain reaction(PCR) methold was developed for detecting duck plague virus.We found a 765-EcoR I fragment which cloned frome the genome of the duck plague vaccine(DP-VAC) virus in the GeneBank.The fragment sequence was similar to the 3’ends of an undefined open read frame and the gene for DNA polymerase protein in other herpesviruses.A set of primers were found to be specific for the DP-VAC virus.The specificity was tested with genome templates from other avian herpesviruses,including those from the chicken (Marek’s Disease Virus,MDV and Infectious Laryngotracheitis VirusJLTV) and the duck(Duck Hepatitis Virus DHV, Pasteurella multocida, Escherichia coli, Salmonella anatum and Riemerella Anatipestifer),but amplicons were not produced.This PCR test is highly specific for duck plague vaccine strain,equivalent to five genome copies.The PCR can detect lOfg of DNA from the duck plague vaccine strain. The test is more 10 times sensitive than tissue culture for detecting duck plague virus.The speed,sensitivity,and specificity of this PCR provide a greatly improved diagnostic and reaction tool for studying the epizootiology of duck plague.In this paper the study on the regularity of duck plague virus vaccine strain distributing in the ducklings was reported. The ducklings were Sichuan Duck with maternal antibody, which have been immunized by different routes(Subcutaneous, Nose dripping and Orally). The results indicated:(2)The DPV DNA can be detected in the heart,liver,spleen, kidney,bursa of fabricius,thymus,pancreas,medulla oblongata,cerebrum,cerebellum from vaccined ducklings 4 hours after subcutaneous injected.8 hours past, all the tissues’s DPV DNA can be found out.(2)The DPV DNA can be detected in tongue and esophagus from ducklings 4 hours past vaccined orally,and be detected in heart,liver,spleen,kidney,thymus,pancrea,medulla oblongata,, cerebrum, cerebellum,tongue, esophagus and blood.(3)There was no DPV DNA in 4 hours after nose dripping route immunized in any tissues of duckling. 8 hours later, the DPV DNA were found out in heart,liver,spleen,kidney,thymus,medulla oblongata,cerebrum, cerebellum,tongue,esophagus and blood.(4)The tissues which be detected the earliest and the highest efficiency were the liver and brain (cerebrum, cerebellum and medulla oblongata).更多还原