Preliminary Study Of H5 And H9 Subtype Avian Influenza Virus Micoarray Technique

Avian influenza(AI) is a infection and /or disease syndrome in both wild bird and poultry, which caused by avian influenza virus type A of the Orthomyxoviridae family. Highly pathogenic avian influenza (HPAI) is classified to"List A"disease by the Office International des Epizooties(OIE). AIV genome contains eight negative, single strand RNA virus, which is divided into sixteen hemagglutinin(HA) and nine neuraminidase(NA) subtypes. Identification of AIV subtypes is mainly based on HA inhibition test and NA inhibition test adopted by WHO.Micoarray is a kind of time saving, high parallel and throughput new technology, which can be applied to the detection of hundreds of disease markers simultaneously. This study was a preliminary exploration of applying micoarray technique to identification of AIV subtypes. This study wants detect the subtypes of AIV through a manipulation on the micoarray platform. The processes include:1. The preparation of the probesHA gene of H1~H15(without H12) subtypes and NA gene of N1~N9 subtypes, NP gene (segmented into three separately), M and NS ; NDV,IBDV,IBV genes and GAPDH gene(positive control)were amplified by PCR. The eighty-five PCR products were precipitated by ethanol,condensed to final concentration above 1μg/μl.2. Preparating and optimizating of the micoarrayAll the probes ,negative and blank controls were spotted onto slides coated by chipwriter. The reaction conditions were optimized from dotting and hybriding conditions. The result indicated that the signal was more idealistic when the dotting concentration was 300~1000μg/μl, the printing buffer was 3×SSC+1.5M batine, the humidity was 60%RH ,the hybridizing temperature was 42℃, and the hybridizing time was 8~12 h.3. Comparation of different sample fluorescence-labeledThe method was that the sample was labelled by Cy3-Unit12 during reverse transcription or labelled by Cy3-dUTP during one step method multiple RT-PCR technique. This study established a universal primer RT-PCR method in the application of micriarray, improved the mixing efficiency of Cy3 and reduced costs.4. Application of the chipIn the study, H5N1 and H9N2 subtypes AIV were examined by the preparated micoarray. The results were accordance with, which proved that the micoarray could identify the subtypes of AIV. Through the sensitivity, specificity, reproducibility and stability testing, this study proved that the micoarray can be used in clinical detection.The results showed that the diagnostic microarray can test these subtypes of AIV, which approved that our established methods were practical. The progress of this research laid solid foundation for the setup of higher density microarray.