| The purpose of this study was to detect avian influenza virus (AIV) subtype H5, H7 and H9. The reverse dot blot hybridization assay and microfluidic chip method for identification of AIV subtype H5, H7 and H9 was established. The sensitivity of microfluidic chip method was compared with that of reverse dot blot hybridization assay.The study consists of three parts:Part I: Cloning and sequence analysis of HA gene of avian influenza virus subtype H5, H7 and H9Two pairs of primers were designed according to the hemagglutinin's conserved sequenses of AIV subtype H5 and H9. The interested fragments were amplified and inserted into a vector pGEM-T. Ligation products were transformed into E.coli competent cells. Doubtful strains were distinguished by blue-white spot screening, and then identified by restrictive endonucleonase, PCR and sequencing. Sequencing results showed that the cloned HA genes were successfully identified for AIV subtype H5 and H9. The recombinant plasmids were named as pGEM-T-H5 and pGEM-T-H9, respectively.Based on the published hemagglutinin genes of AIV subtype H7 A/chicken/Italy/1285/2000(H7N1) (Genbank NO.: CY015014) in Genbank, 208 bp (973~1 180 bp) fragments were artifical synthesized,and then inserted into a pMD18-T vector. The recombinant plasmid was named as pMD18-T-H7。PartⅡ: Development of reverse dot blot hybridization assayOligonucleotide probes and biotinylated primers were designed according to the conserved sequenses of hemagglutinin (HA) genes. The oligonucleotide probes were immobilized onto the positively charged nylon membrane by Ultraviolet (UV) radiation. A multiple RT-PCR was established with biotinylated primers for the amplification of HA fragment. Taget DNA fragments were amplified and then hybridized with oligonucleotide probes on the membrane at 42℃. Color reaction was carried out after hybridization. The presence of clearly visible purple-blue spots on the membrane was considered to be a positive hybridization reaction. The conditions of this assay was optimized, the whole procedure could be completed within 2.5h post-RT-PCR processing. 10 pg RNA of H5 subtype could be detected by reverse dot blot hybridization assay, and the detection limit of H9 subtype was 1pg RNA. 1 pg plasmid DNA of H7 subtype could be detected. The detection sensitivity of RDB was 10-fold higher than agarose gel electrophoresis. No cross-hybridization was detectecd between subtype H5, H7 and H9.After using Rheonix's RT Hyb hybridization solution, the hybridization could be done at room temperature, the detection sensitivity was equivalent with that of 42℃, and the background was lower. Reverse dot blot hybridization assay for identification of AIV subtype H5, H7 and H9 was specific and sensitive, which might become a rapid, effective method for identification of AIV.PartⅢ:Establishment of the microfluidic chip method for detection of avian influenza virus subtype H5, H7 and H9Based on reverse dot blot hybridization assay, the microfluidic chip method for identification of AIV subtype H5, H7 and H9 was established. Nucleic acid extration, PCR reaction and hybridization were integrated on one chip; AIV could be detected rapidly, accurately and automaticly. Verified by conventional methods, the results of microfluidic chip method were reliable. The whole procedure could be completed within 5 h; 1 pg RNA of H9 subtype could be detected by microfluidic chip method, the detection limit of H7 subtype was 1 pg plasmid DNA, and 10 pg plasmid DNA of H5 subtype could be detected. The detection sensitivity was equivalent with that of reverse dot blot hybridization.The microfluidic chip method used fewer reagents, worked without special equipments under a higher efficiency. This assay will provide simple and effective technical support for rapid discrimination of AIV subtype H5, H7 and H9.
|
PDF Full Text Request