| 区分禽流感病毒亚型诊断基因芯片的构建 |
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| 引用本文： | 杨忠苹,王秀荣,石霖,田丽娜,王煜,陶启蒙,陈化兰.区分禽流感病毒亚型诊断基因芯片的构建[J].中国动物检疫,2008,25(10):29-32. |
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| 作者姓名： | 杨忠苹 王秀荣 石霖 田丽娜 王煜 陶启蒙 陈化兰 |
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| 作者单位： | 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 |
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| 基金项目： | 国家科技攻关计划，国家自然科学基金 |
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| 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。
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| 关 键 词： | 禽流感 基因芯片 亚型鉴别 |
Fabrication of Diagnostic microarray for Subtyping Avian Influenza Virus |
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| YANNG Zhongping,WANG Xiurong,SHI Lin,TIAN Linn,WANG yu,TAO Qinmeng,CHEN Hualan.Fabrication of Diagnostic microarray for Subtyping Avian Influenza Virus[J].China Journal Of Animal Quarantine,2008,25(10):29-32. |
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| Authors: | YANNG Zhongping WANG Xiurong SHI Lin TIAN Linn WANG yu TAO Qinmeng CHEN Hualan |
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| Institution: | 1(1.Animal Influenza Laboratory of Ministry of Agriculture and National Key Laboratory of Veterinary Biotechnology of Harbin Veterinary Research Institute of CAAS, Harbin, 150001;2.Chongqing Entry Exit Inspection and Quarantion Bureau,Chongqing,400020) |
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| Abstract: | To develop the diagnostic microarray for detection of the AIV haemagglutinin subtypes H5,H7 and H9 and neuraminidase subtypes 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 0.3μg/μl and spotted onto aldehyde glass slide with microarray printing system.The spot space was 450μm .The diameter of spot was 220μm .The spotted slide was dried at room temperature for at least 24h,hydrated for 10s at 65℃.UV cross-linking was carried out by placing the slides, in a crosslinker, exposing the slides to 65mJ of energy.After blocking and denaturation,the diagnostic 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. |
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| Keywords: | influenza Diagnostic microarray Subtyping |
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