Development Of Two-step H5-RT-LAMP System For Rapid Diagnosis Of H5 Avian Influenza Viruses And Investigate The Molecular Epidemiology Of H5-subtpye Avian Influenza Virus In Sichuan Province

As generally known,Avian influenza is caused by Influenza A viruses,which normally lead multi-syndrome to the domestic birds and waterfowl poultries.There were some sporadically reports about human infection with Influenza A viruses,such as H5N1 and H1N1.Based on the virulence of different influenza A viruses,they can be divided into three gourps,containing High Pathogenicity Avian influenza(HPAI),Low Pathogenicity Avian influenza(LPAI)and None Pathogeicity Avian influenza.Influenza virus belongs to Orthomyxoviridaes famliy,Influenza virus sub-family,which is a negative RNA virus containing 7-8 segments.In this study,A two-step H5-Reverse transcript loop-mediated isothermal amplification(Two-step H5-RT-LAMP)assay was conducted to detect H5 avian influenza virus infection in this research.Six primers were specially designed for recognizing eight distinct fragments of the HA gene,which could be made available for highly sensitive and specificity,less reaction time and temperature conditions were optimized to be 30 min at 63 ?.The protocol also did any cross-react with other subtype avian influenza virus(H7 and H9),goose parvovirus,muscovy duck virus and duck plauge virus,and there was amplicons could be detected when we used the H5 subtype avian influenza viruses as template.The detection limits of vivo transcription HA gene through the Two-step H5-RT-LAMP was just 10 copies while the limitation of RT-PCR was around 1000 copies.Therefore,we belive this protocol could be regarded as a pre-screen tool for identifying H5 subtype avian influenza viruses from clinical waterfowl samples.Between 2010 and 2012,we collected 500 suspected samples from Sichuan Province.Then we used the WH0-RT-PCR to detect these samples.In this research,we identified five epidemic strains,named Zigong strain?Deyang strain?Chengdu strain?Mianyang strain and Yaan strain.Hemagglutinin protein not only being the surface protein and major surface antigens,it also plays an important role of host specificity amd virulence.So we amplified the HA gene of two strains,then cloned and sequencing.After that,we carried out bioinformatic research on the amino acid coded by HA gene.The results indicated that receptor binding site of the above five strains are both QSG;the cleavage site of the HA gene were PQRERRRKR/GLF and PLREKRRRKR/GLF,which mode was regarded as the molecular characterization of high pathogenicity.Based on the Evolutionary tree of HA gene constructed by Mega 4.0,the five strains showed more closely related to each other,which were all divided into clade2..