A Novel Foundation Of Detecting Avian Influenza Virus By Microfluidic Chip And Encoded-microsphere

Avian influenza(AI) is a number of influenza A virus and can cause fatal disease for avian specises. HPAIV was placed the class A infectious disease by OIE. Since, It was reported in 1878 and has beening happened all over the world. It has not only led to the seriously loss of economy of the poultryenterprise all over the world, but also threated human health seriously. Avian Influenza Virus(AIV) H5N1 in 1997, H9N2 in 1999, H7N7 in 2003 and H5N1 in 2004 infected human beings successively so that people understand the public sanitation significance of avian influenza.High polymer microsphere is point to solid particle that diameter ranged from micron to nanometer, loaded fluorescent matter and be able to stimulate fluorescence when accepted energy stimulation outside. Newly developed high polymer microsphere was a protein carrier with good capability, characterized by homogeneous gain size, stable and capable of coupling with biological activated materials for multiparametric analysis. Due to they can absorb antibodies for analyte corresponding antigens, it always apply to immunoassay diagnostic agent or ingredient of diagnostic kits. And Flow cytometry is a general method for analyzing microparticles such as cells, bacteria and other microorganism with high efficiency, integrating optics, fluidics, electronics, and computer technology. This research is on the base of traditional sandwich ELISA, and use the polystyrene microsphere as protein carriers, combination of immunofluorescence technique, flow cytometric and so on. In chapter 2, Based on preparation and purification of the polyclonal antibody, a novel immunoassay of detecting the Avian influenza vius, by cytometry with flowing fluorescence-coded microsphere as protein carrier was studied and in coupled with flow cytometry, traditional ELISA technique and fluorescent antibody labeling technique. The research on sensitive, stable and specific experimentation, and constrast this method to traditional sandwich ELISA about sensibility. Under experiment verification, the method was simple, fast, specific and stable. Sensibility of this method is the same as traditional sandwich EL1SA when detect AIV. The method was applied to detect chicken swab samples of which included 30 from Xinsheng farm of Beijing Chaoyang district, The negative and positive control are correct, the results are compared with sandwich ELISA, both of them are negative for 30 samples。Microfluidic Chip technology is the foreland of analysis that nineties developed last century, but provides on kind of be simple,fast,accurate analysis method. In coupled with flow cytometry, Makes use of microsphere that asticted with sheathing fluid to line up into a column and move one by one, having provided for theory to counting capture signal in detecting area, providing new idea for realizing quick diagnosis of epidemic disease. In part two, Microfluidic Chip is applied to the detecting mode, Using Na₂PO₄ solution added with hydroxypropyl methyl cellulose (HPMC) as buffer and sheathing fluid. The method realized fast detection for AIV.The research on reagent and sample is little and cheap, it also set the foundation for the research of detecting multiple animal diseases simultaneously.