鸡黄病毒RT-PCR检测方法的建立

为建立鸡黄病毒(CFV)分子生物学检测方法,本研究以虫媒介黄病毒基因3’末端通用引物EMF1/VD8作为鉴别引物,以CFV分离株CJD-05为模板,建立了检测CFV的RT-PCR方法。RT-PCR扩增产物测序结果显示,其扩增片段为708 bp,而该方法对其它禽类病毒病原核酸的扩增结果均为阴性,表明该方法具有较高的特异性。扩增的特异性片段与GenBank中CFV、鸭黄病毒和鹅黄病毒序列同源性达到100%、98%和99%。对参考病毒进行梯度稀释检测,该方法检测CFV的敏感度可达10-3TCID50/0.1 mL。采用所建立的RT-PCR方法对2009年福建省部分鸡场的112份临床样品进行检测,结果显示37份为CFV阳性。本研究建立的RT-PCR方法可以作为CFV在临床检测和分子流行病学调查的一种检测方法。

Establishment of RT-PCR for detection of chicken flavivirus

To establish a RT-PCR based detection for chicken flavivirus(CFV) infection,,a pair of arthropod-transmitted flavivirus universal primers of EMF1 and VD8 in accordance with 3’ terminal sequence was used to amplify target sequence from the RNA of CFV,and a special fragment of 708 bp was amplified from CFV CJD-05 with a detection limit of 10-3 TCID50,but no amplification was found from other related avian viruses.Sequence analysis showed that the amplified 3’ terminal fragment shared 100%,98%,99% identity with the CFV,duck flavivirus and goose flavivirus,respectively.The RT-PCR was applied to detect 112 clinical samples from several chicken farms of Fujian province in 2009,and 33%(37/112) samples were CFV-positive.The result showed that the RT-PCR technique provided a useful method for clinical diagnosis and epidemiologic survey of CFV.