Ⅰ型鸭肝炎病毒逆转录套式PCR检测方法的建立  

Establishment of a nested RT-PCR assay for detection of duck hepatitis virus type Ⅰ

作　　者：黄显明[1] 张小飞[2] 李春芬[1] 廖俊伟[1] 毛火云[1] 

机构地区：[1]安徽农业大学动物科技学院 [2]江苏省农业科学院兽医研究所

出　　处：《中国兽医科学》2008年第1期25-28,共4页Chinese Veterinary Science

摘　　要：根据GenBank中登录的Ⅰ型鸭肝炎病毒(DHV-Ⅰ)A66株的RNA聚合酶基因序列,设计合成了2对引物,建立了适合DHV-Ⅰ快速检测的逆转录套式PCR方法(RT-nested-PCR),采用该方法对DHV-ⅠA66弱毒株和R85952强毒株进行了检测。结果显示,均能扩增到304 bp的条带,而正常鸭胚、健康鸭肝组织、鸭瘟病毒、鹅细小病毒、禽流感病毒(H9亚型)、新城疫病毒、传染性腔上囊病病毒、减蛋综合征病毒、鸭源大肠杆菌、鸭疫里氏杆菌和鸭源多杀性巴氏杆菌的扩增结果均为阴性。该方法第1次扩增的敏感性是100 pg,第2次扩增的敏感性是1 fg,第2次比第1次扩增的敏感性高105倍。表明,所建立的逆转录套式PCR方法可用于鸭病毒性肝炎(DVH)的临床诊断、病料检测和分子流行病学调查等。According to the sequence of RNA polymerase gene of duck hepatitis virus type Ⅰ （DHV-Ⅰ ） A66 strain published in GenBank, two pairs of primers were designed and synthesized. A nested RT- PCR assay for rapid detection of DHV-Ⅰ was established. A specific 304 bp fragment was amplified from RNA templates of DHV- Ⅰ A66 attenuated strain and R85952 virulence strain, but no bands were amplified with templates extracted respectively from normal duck embryo, healthy duck liver tissue, duck plague virus （DPV）, gosling parvovirus （GPV）, avian influenza virus （AIV） subtype Hg, Newcastle disease virus （NDV）, infectious bursal disease virus （IBDV）, egg drop syndrome virus （EDSV）, Escherichia coli （E. coli） of duck origin, Riemerella anatipestifer（RA）, and Pasteurella multocida （PM） of duck origin. Sensitivity of the 1st and 2nd amplifications by the nested RT-PCR assay was 100 pg and 1 fg, respectively. The sensitivity of the 2nd amplifications was increased by l0S times. The results showed that the nested RT-PCR assay could be used as a method for the diagnosis and detection of clinical cases, and for molecular epidemiological investigation of DVH.

关 键 词：Ⅰ型鸭肝炎病毒 逆转录套式PCR 检测 

分 类 号：S852.659.6[农业科学—基础兽医学] Q503[农业科学—兽医学]