区分禽流感病毒亚型诊断基因芯片的构建  

Fabrication of Diagnostic microarray for Subtyping Avian Influenza Virus

作　　者：杨忠苹[1] 王秀荣[1] 石霖[1] 田丽娜[1] 王煜[2] 陶启蒙[1] 陈化兰[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所农业部动物流感重点开放实验室兽医生物国家重点开放实验室,哈尔滨150001 [2]重庆出入境检验检疫局,重庆400020

出　　处：《中国动物检疫》2008年第10期29-32,共4页China Animal Health Inspection

基　　金："十五"国家科技攻关;不同动物流感病毒免疫学及高通量检测技术的研究和开发(2004BA519A23);国际合作;防禽流感高效生物制剂试生产开发;编号(20072326)

摘　　要：制备了可同时区分AIVH5、H7、H9血凝素亚型及N1、N2神经氨酸酶亚型的基因诊断芯片。试验以重组质粒为模板,进行PCR扩增,然后用异丙醇沉淀法进行纯化,制备探针及内参基因。将探针及内参基因用点样缓冲液稀释到0.3μg/μL后用芯片点样仪将其点制到醛基化基片上,样点中心间距450μm,样点直径220μm。将点好的基片在室温干燥24h以上,后经65℃再水合10s、80℃干燥、65mj紫外线交联照射25min后,再用封闭液封闭,95℃变性处理,成功构建了能区分禽流感血凝素H5,H7,H9亚型及神经氨酸酶N1,N2亚型的检测基因芯片。在PCR扩增中标记待检样品,对制备的检测芯片进行质量检验,结果表明,制备的区分禽流感病毒亚型基因芯片可区分AIV部分亚型。To develop the and neuraminidase subtypes diagnostic microarray for detection of the AIV haemagglutinin subtypes H5,H7 and H9 N1 and N2 the probe genes of H5,H7,H9,N1,N2 and control gene of GAPDH were amplified by PCR with recombinant plasmids templates respectively and precipitated by isopropanol. They were diluted to 0.31ug/ul and spotted onto aldehyde glass slide with microarray printing system.The spot space was 450um .The diameter of spot was 220um .The spotted 65℃.UV cross-linking was carried out gy.After blocking and denaturation,the by slide was dried at room temperature for at least 24h,hydrated for 10s at placing the slides, in a crosslinker, exposing the slides to 65mJ of enerdiagnostic gene chip for detecting H5,H7,H9,N1,N2 were produced successfully.Prepared samples were labelled by PCR with fluorescence CY3 for testing its qualification.The results of qualification test showed that the diagnostics are qulificated and can be used to detect some subtypes of avian influenza virus at the same time.

关 键 词：禽流感 基因芯片 亚型鉴别 

分 类 号：S852.659.5[农业科学—基础兽医学]