Development And Application Of Low Density DNAarray For Diagnosing The Subtypes Of SIV

Swine Influenza is an acute, high contagiousness respiratory disease of swine with a type A Influenza virus which belongs to the Orthomyxoviridae family. At present, eight subtypes of Swine Influenza Virus(SIV) which have been isolated are H1N1, H1N2, H1N7, H3N2, H3N6, H4N6, H9N2 and H5N1, the frequently used methods for identifying subgroups Influenza virus are hemagglutination inhibition assay(HI) and neuramidinase inhibition assay (NI). Besides, the agar diffusion reaction, enzyme linked immunosorbent assay and other serological methods can identify subgroups Influenza virus. Whereas these methods are all lower sensitivtive and hadro-dependence by experience. PCR is a quick and precise method, while the vukgar PCR only test one subtype once. Therefore it can not satisfy the need of detection.This research constructs low density DNAarray for diagnosing the subtypes of SIV by using the technology of DNAarray, multiplicitas PCR and nucleinic acid mark, which can diagnose the subtypes of SIV and the type A Influenza virus at the same time. Major studies are processed as follows:Part 1: Establishment of Low Density DNAarray for Diagnosing the Subtypes of SIV This part includes probe concentration options, biotin-11-dUTP concentration options, hybridization temperature and hybridization time options, sensitivity, specificity, reproducibility and constant testing.The hemagglutinin(HA) and neuraminidase(NA) gene of five subtypes influenza A virus were amplified by RT-PCR with primers based on the diversity of HA and NA gene. Meanwhile, conserved cDNA fragments of M were amplified by RT-PCR with consensus primer. Each fragment was cloned in pMD18-T Simple Vectors to construct recombinant plasmid respectively, and constructed recombinant plasmid for DNA probe preparation and recovered DNA probes were spotted on located sites on nitrocellulose filter to be DNAarray. After sequencing, the result was tested by using BLAST and compared with the corresponding subetypes. Aliment reports show that the sequence is above 90% with the bulk corresponding subetypes. After purificating, the probes are diluted to 75 ng/μ, and marked nitrocellulose filter, fixed to two hours, determined the preparation of genechip and condition of hybridization. The results show that the Biotin-11-dUTP is 80μM, probe is 75 ng/μl; hybridization temperature is 42℃, hybridization time is 1 hour. These DNAarrays have specificity and good reproducitivity. The sensitivity of genechip is one dilution higher than PCR, and two dilution higher than viral isolation. These DNAarray provids a quick, sensitive and high-flux method for large-scale epidemiology investigation and quarantine of Swine Influenza Virus.Part 2: Application of dissymmetry PCR in DNAarrayDissymmetry PCR can be applied to prepare single strand PCR products, and that can reduce the lose of self-hybridization in DNAarray hybridization. It raises the hybridization rate, and reduces the final prducts of PCR. This research chooses 1:25, 1:50, 1:100 and 1:200 titre. It shows that the optimization primer concentration is 1:100, the sensitivity is raised 10～100 times comparing with the conventional PCR.Part 3: The primary application of DNAarray in diagnosing the subtypes of SIVThe DNAarray is applied to detect the nose swab or tissue samples of health swine or illness swine from Qingdao,Yantai,Weifang,Jining,Nanchang,Huludao and so on. The result shows that the coincidence of DNAarray and PCR is above 87%, but it has significant diversity with the method in which the virus was isolated by embryo passage. It shows that this DNAarray can be applied to monitor the swine influenza, the sensitivity is higher than PCR.