Primary Study Of Gene Chip For Detecting Influenza Viruses And New Castle Disease Virus

Avian influenza and New castle disease are both the potent infectious diseases in poultry,causing great harm to the poultry industry and economic development. They are often mixed infection clinically, and the symptoms of the disease are similar, which have brought great trouble to the differential diagnosis .the key of the prevetion and control is the rapid diagnosis in an early stage, which have a direct effect on continued development of China's poultry industry and people's health. At present, The main methods for detection of Avian influenza and New castle disease virus contain virus isolation, serological testing, the influenza virus antigen test and multiple PCR detection methods. However, neither of them precisely classifies the multiple types or subtypes of influenza virus. And that it is necessary to find an efficient, high-throughput and rapid methods for detection and typing of influenza and New Castle Disease virus. Gene-chip technology has broad application prospects for rapid test for the differential diagnosis . It is a cross-cutting-edge technology integrated with biology, physics, chemistry, computer science and micro-electronics integration. It is not only fast, sensitive, specific, high-throughput automation, but also analyze the data and determine the outcomes based on computer software to enhance reliability of the results. Gene chip technology provides a possible way for the detection and typing of the Avian influenza virus and New Castle Disease virus at the same time. Therefore, this study on the part of the avian influenza HA, NA and New Castle Disease virus F gene Cloning and sequencing, and on the designing specific oligonucleotide probes for gene chip detecting multi-subtype avian influenza viruses and New Castle Disease virus on the basis of an gene sequence analysis, has far-reaching significance for early differential diagnosis and purification of New Castle Disease virus and avian influenza virus subtypes.First of all, this study using RT-PCR method to obtain the influenza virus for HA and NA gene sequence including the non-coding region,that contain A / New Caledonia/20/99 (H1N1), A/Swine/GuangDong/1/2003 (H3N2), A/chinken/JL/9/2004 (H5N1), AIV Isolate3 (H9N2),have obtained NDV (CC strain, F48E9 strain) F gene-containing DNA fragment, that has been sequenced; through synthetic; we won the HA gene fragment of the H7 subtype and complete ORF nucleotide sequence; A comparative study on HA and NA genes viruses appearing in different countries at different times,has determined homology relationship between 16 HA and 9 NA subtypes of influenza virus. Results: 16 HA subtypes genes homology rate of influenza virus are between 49.3 percent and 74.5 percent 9 NA homology rate subtypes genes homology rate of influenza virus are between 50.4 percent and 67.9 percent; homology rate between the cloned gene compared with the same subtype of its corresponding sequence are between 80percent and 97.9 percent.Those lay the foundation for a follow-up application of important genes, multiple PCR primer and specific oligonucleotide probes.Secondly,According to the results of comparative sequence analysis We design 8 pairs of Multiple PCR primers testing HA gene and NA gene of influenza virus and New Castle Disease virus F gene, 16 pairs of specific oligonucleotide probes, one pair of positive probe, and one pair of probe for post-processing using bioinformatics software At the same time, manufacturing process of the gene chip has been optimized, determining the humidity, concentration,buffer, Washing Process, preconditioning procedure. The results showed that the best humidity of application of sample was 50 percent,the best Concentration was 50μM,CapitalBio gene chip buffer dissolved probe got better results. We used 37℃hydration overnight after treatment,then fixed and washed. Preconditioning procedure before hybrid cleaning in 42℃containing0.5 percent SDS for 10 min, in 42℃distilled water for 4 min,in dehydrated alcohol for 2min,can decrease chip background in effect.Thirdly, This study prepared fluorescent sample testing positive with The established multi-PCR system. through changeing an important factor that affects the efficiency of the hybrid one by one, We optimized the conditions for hybridization Chips.We find that For the probe the hybrid is better in 42℃than other temperature and it will gain a better signal of hybridization for 5h. The best selection of experimental data, we have successfully completed a gene chip to optimize the conditions for hybridization.Finally, We tesed HA gene of H1,H3,H5,H7,H9 subtype,NA gene of N1,N2 subtype influenza viruses and the F gene of New Castle Disease virus with the prepared chip. The results showed that the chip-specific prepared by the research was good. On the negative control, No signal was detected. Sensitivity results showed that Sample size determination of 1×10~3 copies /μl for the detection line. The study has developed initial detection chip of influenza virus HA gene,NA gene and New Castle Disease virus. After testing, the chip is accurate, specific, rapid and sensitive, safe and easy to operate.It is of great significance of early monitoring and timely prevention and control,and there are good prospects for development...