鸭黄病毒感染逆转录半套式PCR检测方法的建立  

Development of a semi-nested RT-PCR assay for detection of duck flavivirus

作　　者：唐熠[1] 刁有祥[1] 高绪慧[1] 于春梅[1] 张大丙[2] 岳澄滨 

机构地区：[1]山东农业大学动物医学院,山东泰安271018 [2]中国农业大学动物医学院,北京100193 [3]潍坊乐港食品股份有限公司,山东昌乐262411

出　　处：《中国兽医学报》2012年第4期517-520,共4页Chinese Journal of Veterinary Science

基　　金：现代农业产业技术体系建设专项资金资助项目(nycytx-45-11)

摘　　要：根据GenBank发布的黄病毒Bagaza毒株的NS3基因的保守序列设计了3条引物,建立了一种适用于鸭黄病毒的半套式PCR快速检测方法。采用该方法对鸭黄病毒山东分离株进行检测,结果半套式PCR对2株分离株均能扩增出277bp的特异性条带,而对鸭瘟病毒、禽流感病毒(H9亚型)、新城疫病毒、传染性法氏囊病病毒、减蛋综合征病毒的扩增结果均为阴性;经检测该方法第1次扩增的敏感性为1×105拷贝/μL,第2次扩增的敏感性为1×102拷贝/μL,第2次扩增的敏感性比第1次高103倍;该方法对山东省各地采集的24份疑似病料可检出22份阳性。表明本试验所建立的套式PCR方法可用于鸭黄病毒感染的临床诊断和流行病学调查。According to the sequence of NS3 gene of flavivirus Bagaza strain published in GenBank,three primers were designed and synthesized.A semi-nested RT-PCR assay for rapid detection of duck flavivirus was established.A specific 277 bp fragment was amplified from RNA templates of flavivirus of Shandong isolates,but no bands were amplified with templates extracted respectively from duck plagues virus（DPV）,avian influenza virus（AIV） subtype H9,Newcastle disease virus（NDV）,infectious bursal disease virus（IBDV）,egg drop syndrome virus（EDS76）.Sensitivity of the amplifications by the semi-nested RT-PCR assay was 1×105 copies/μL and 1× 102 copies/μL,respectively.The sensitivity of the 2nd amplifications was increased by 103 times and the had a high detection rate of clinical samples.These results suggested that the semi-nested RT-PCR assay could be used as a method for the diagnosis and detection of clinical cases,and molecular epidemiological investigation of duck flavivirus.

关 键 词：鸭黄病毒 NS3基因 逆转录半套式PCR 

分 类 号：S852.65[农业科学—基础兽医学]