摘要
本研究根据GENBANK中查出的禽流感病毒H_5亚型，禽流感病毒H_9亚型的HA片段基因组序列，利用DNASIS软件分别对AIV的H_5、H_9亚型HA基因区域进行同源性比较，设计筛选出两对联合PCR反应的特异性引物，其中一对是H_9亚型通用引物，扩增出HA片段695bP，另一对引物为H_5亚型的通用引物，扩增出的HA区域部分目的片段约为448bP。建立各自的单项RT-PCR，并用电泳鉴定PCR扩增产物，结果证明为各自的特异性核酸片段。在单项RT-PCR基础上，优化引物浓度，Mgcl_2浓度，Tag酶、dNTP浓度，循环次数等反应条件后，建立了H_5、H_9亚型的多重RT-PCR，同时扩增出禽流感病毒H_5、H_9两种亚型不同大小的特异性片段，并进行了反应条件的优化。建立的多重RT-PCR扩增其它相关病毒未见阳性结果。两个亚型的单项RT-PCR可以检测鸡胚尿囊液培养物10~(-3)稀释物，多重PCR可以检测到10~(-2)稀释物。初步应用四份子鸡胚尿囊液培养物，六份临床病料的检测，并与电镜负染、琼扩及病毒分离的结果进行比较，此诊断方法与病毒分离的结果一致，比琼扩和电镜负染检出率高，可用于临床和试验室诊断。
A multiplex reverse transcriptase-polymerase chain reaction was optimized to Simultaneously detect H_(5) H_(9) subtype for Avian Influenza virus . The hemaglutinin (HA) of AIV plays the key role in determing the pathogenicity ,cell receptor binding property and host range of the virus. The homology of The HA sequences reported and registered in Genbank of different strains of H_(5). H_(9) subtype was respectively analyzed and compared with each other .The conservative domin of HA seqence of H_(5), H_(9) subtype was selected for PCR amplification .Two sets of specific primers were designed . H_(5) subtype can be amplified 448bp .H_(9) subtype can be amplified 695 bp .
The designed primers were use to PCR reaction . The result showed that the design of the two sets of primers was right .Then the optimal conditions of PCR for each virus were determined by orthongonal assay, two sets of primers were combined and used for ampliation trials. The rudimentary application of the multipex RT-PCR has the same results with Seprate Cultivation of the virus, the positive results acqired is higher then AG and EM.
The method described is very practable ,sensitive,specific,simple. It could be used in diagnosis of HS. Hg subtype of AIV.
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