禽流感病毒三种检测方法的比较研究

Comparison of Three Methods for the Detection of Avian Influenza Virus

对分别应用病毒分离鉴定、反转录-聚合酶链式反应(RT-PCR)、间接免疫荧光(IFA)3种方法对病料组织中的禽流感病毒(AIV) 进行检测与比较研究。结果显示:经鸡胚尿囊腔接种分离到的病毒,通过用禽流感病毒H5、H9亚型单克隆抗体所进行的血凝(HA)和血凝抑制试验(HI),鉴定为AIV H5亚型毒株;应用RT-PCR技术直接对病料组织抽提的RNA样品进行检测,从病料组织能扩增出大小为229bp的AIV通用目的片段和大小为380bp的H5亚型特异性片段;取病料组织的过滤液接种狗肾上皮细胞(MDCK)单层,24h后用H5亚型AIV特异性的单克隆抗体进行间接免疫荧光试验,结果在细胞核、细胞质内观察到特异性的荧光。研究的结果表明,3种技术都可对病料样品中的AIV进行检测,而RT-PCR和IFA两种方法还可直接对病料样品中的病毒进行亚型的鉴定。相对而言,后两者具有更快速、更敏感、操作更简便的特点。

Three methods including the classical virus isolation and identification, reverse transcriptase polymerase chain reaction （RT-PCR） and indirect fluorescence assay （IFA） were used to detect avian influenza virus （AIV） in the sample and were compared. A virus was isolated by egg allantoic cavity inoculation and identified as H5 subtype of AIV with a monoclonal antibody （McAb） against H5 subtype via the hemoagglutination （HA） and hemoagglutination inhibit （HI） test. RT-PCR was performed to detect the sample directly and a 229bp-band of AIV specific and a 380bp-band of H5 subtype specific were both amplified with the designed primers. The tissue suspension was infected onto the monolayer of Madin-Darby canine kidney （MDCK） cells and a H5-McAb was applied to run the IFA on the monolayer cells at 24 hours post-infection. The fluorescence in both nucleolus and cytoplasm were observed. The results of the study demonstrated that these three methods could be applied to detect the AIV in the sample, while RT-PCR and IFA could identify the subtype of AIV at the same time, and have the advantages over the classical method in the aspects of sensitivity, rapidity and simplicity.