Avian Influenza Virus H-<sub> 5 </ Sub> H <sub> 9 </ Sub> Subtype Of Multiple Rt-pcr Method For Detection And Preliminary Applications

A multiplex reverse transcriptase-polymerase chain reaction was optimized to Simultaneously detect H₅ H₉ 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 H₅. H₉ subtype was respectively analyzed and compared with each other .The conservative domin of HA seqence of H₅, H₉ subtype was selected for PCR amplification .Two sets of specific primers were designed . H₅ subtype can be amplified 448bp .H₉ 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.