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Abstract
The aim of this project was to develop three diagnostic systems: a DNA chip diagnostic system, a multiplex RT-PCR diagnostic system for H5N1 virus subtype, and a real-time RT-PCR diagnostic system for influenza virus type A. The DNA chip diagnostic system was developed for differential diagnosis of some important swine and avian influenza virus subtypes H1, H3, H5, H6, H7, H9 and N1. Eight sets of primers and ten specific probes were designed based on H1, H3, H5, H6, H7, H9, M and N1 gene sequences of influenza virus type A. The specific genomic regions of the pathogens were amplified by three sets of the multiplex RT-PCR and then were subjected to dot blot hybridization with specific oligonucleotide probes which had fixed onto the chips. A prototype of DNA-chip diagnostic system for some important subtypes of swine and avian influenza viruses was also developed. The results showed that the DNA-chip diagnostic system could differentiate the H1, H3, H5, H6, H7, H9, M and N1 subtypes of influenza viruses. Three sets of primers of the multiplex RT-PCR diagnostic system for H5N1 virus subtype were designed to amplify the M、H5 and N genes of avian influenza viruses. The results showed that the multiplex RT-PCR could amplify the expected genomic regions of H5N1 virus subtype and showed three visible bands of RT-PCR products in agarose gel. The real-time RT-PCR diagnostic system also was established for the detection of influenza virus type A. The results showed that the real-time RT-PCR could detect 15 subtypes, H1 to H15, of influenza virus type A.
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