The Study Of H6Subtype Avian Influenza Virus Pathogenic Surveillance And Rapid Diagnosis Method

The H6subtype avian influenza virus (AIV) belong to low pathogenic influenza A virus, waterfowl and wild birds is the main host of the H6subtype AIV. H6subtypes AIV can also infected mammal. Such as rats and mink, The report says even can detected the antibodies of H6subtypes AIV from healthy young people. H6subtypes AIV may provide genes for highly pathogenic avian influenza virus. Thus may cause a new round of pandemic influenza.Separation H6subtypes AIV From the sample which collected from the live poultry market in the years2009to2011. HI test of preliminary test, RT-PCR Methods for further validation testing. Then do the gene sequencing of the NA gene. From2009to2011get7sample of H6subtypes of AIV,they are one strain of H6N1,two strains of H6N2,one strain of H6N5,two strains of H6N6,one strain of H6N8.Do the gene sequencing for the eight gene fragments of the seven strains H6subtypes AIV separation. After the completion of the seven H6subtype AIV gene sequencing, sequence analysis results on the GENBANK sequence alignment analysis. The results show that, all the8gene segments are of the seven strains H6subtypes AIV separation attributed to the Eurasian lineage, the genome composition might be complicated, the amino acid motif of cleavage site between HA1and HA2was P-Q-I-E-T-R-G, which was the typical characteristics of the LPAIVs.To develop a method of a reverse transcription loop-mediated isothermal amplification(RT-LAMP) assay The viral RNA was amplified with one-step RT-LAMP method, and the reaction conditions were optimized. The results showed that the specificity of the assay was fine without amplification of other respiratory tract pathogenes, and the detection limit of the RT-LAMP assay was0.01pg. The assay is a sensitive, simple,rapid and practical method for detection of H6subtype avian influenza virus in the field. In addition,develop a method of a SYBR Green I fluorescent based real-time RT-PCR (RRT-PCR) to quantify the H6subtype AIV for rapid diagnosis. The results showed that, The detection limit of RRT-PCR was1.07×101plasmid copies, Compared with RT-PCR, more than1000times more sensitivity. The specificity of the assay was high without amplification of the other viruses. The assay is rapid, from the sample processing to report results only3.5h.Comparison the sensitivity of four rapid detected method of H6subtype AIV by RT-PCR, nested RT-PCR, real time PCR and RT-LAMP. The result shows that,between the four method, The highest of sensitivity method is the RRT-PCR, Second for RT-LAMP and nested RT-PCR, PCR is the lowest sensitivity method. Clinical test according to the conditions and demand to choose suitable detection method. A variety of fast detection method combines can better confirm the diagnosis.