流感及H5N1亚型禽流感病毒液相检测芯片的制备

目的 建立利用液相芯片技术检测甲、乙型流感和H5N1亚型高致病性禽流感病毒的方法,并对该方法进行评价。方法 对GenBank中甲型流感病毒的NP、乙型流感病毒的HA以及高致病性禽流感病毒（H5N1）的H5、N1基因片段序列进行同源性比对,根据保守序列,设计针对各基因的简并引物和寡核苷酸探针,制备探针偶联微球,将样本核酸多重PCR扩增产物与微球进行杂交,以Bio-Plex液相芯片检测系统进行芯片检测。结果 该方法可以对甲型流感病毒的NP基因、乙型流感病毒的HA基因以及高致病性禽流感病毒（H5N1）的H5、N1基因同时进行检测,病毒核酸的最低检出量为1pg,检测特异性高。结论 成功构建了甲、乙型流感病毒和H5N1亚型高致病性禽流感病毒液相芯片检测系统,为流感、禽流感的快速检测、诊断奠定了基础。

Establish A Liquid Chip Hybridization Method for the Detection of Influenza Viruses Type A and B and Avian Influenza Virus Subtype H5N1

Objective To establish a liquid chip hybridization method for the detection of influenza viruses type A and B and avian influenza virus subtype H5N1..Methods Four pairs of primers and their corresponding oligonucleotide probes were designed and synthesized according to the H5 and N1 gene from the highly pathogenic avian influenza virus（H5N1）.NP genes from influenza virus type A and HA gene from influenza virus type B had been reported on GenBank.Subsequently,probe-conjugated microspheres were prepared.The mRT-PCR products were hybridized with the oligonucleotide probes conjugated to microspheres and samples were detected using a Luminex instrument.Results The NP gene from influenza virus type A,HA gene from influenza virus type B,H5 and N1 genes from H5N1 could be detected simultaneously.The lowest detection limit with this method was 1 pg of viral RNA,and the specificity of this method was very high.Conclusions A liquid chip hybridization method was successfully established for the detection of influenza viruses type A,B and also the highly pathogenic avian influenza virus subtype H5N1.This method can be used for the rapid detection and diagnosis of influenza and avian influenza infection.