三种病毒性出血热荧光与可视生化基因芯片检测方法的建立-预防兽医学专业毕业论文.docx

基因芯片在DNA检测中大量被使用。 基因芯片在DNA检测中大量被使用。 本研究的目的是建立CCHFV、RVFV和LAV的荧光基因芯片和可视化 基因芯片检测方法。分别以CCHFV和RVFV的S基因，LAV的部分GPC 基因和NP基因，设计3对引物与6条寡核苷酸探针，下游引物分别修饰荧 光素和生物素。以pMDl8T-simple为载体，构建含三种病毒保守序列的重 组质粒pMD—CCHFV、pMD—RVFV、pMD—LAV。以人工合成的保守基因序 列为模板体外转录成病毒RNA作为检测样本。使用多重PCR对目的片段 进行扩增及标记。通过荧光显微镜获取荧光基因芯片杂交结果。标记生物 素的扩增产物与基因芯片杂交后，加入链霉亲和素标记的纳米金，形成生 物素．链霉亲和素．纳米金复合物，再通过银增强试验对信号进行再次放大。 最后通过特异性试验、灵敏性试验及重复性试验对两种基因芯片进行评价。 本研究成功建立了三种出血热病毒的荧光基因芯片和可视化基因芯片 检测方法，且特异性和重复性良好。运用荧光基因芯片可检测出2．1×104 ng／ml的pMD—CCHFV扩增产物，2．82x 1 0‘2 ng／ml的pMD—RVFV扩增产物， 1．73x10。4 ng／ml的pMD．LAV扩增产物。运用可视化基因芯片检测出2．1x10。3 pg／ml的pMD-CCHFV扩增产物，2．82x 1 0。1 pg／ml的pMD—RVFV扩增产物， 1．73x10。2pg／ml的pMD．LAV扩增产物。相比荧光基因芯片，可视化基因芯 片的灵敏度分别提高了100倍(CC耶V)，100倍(R、腰V)，10倍(LAV)。 结果表明，本研究所建立的两种基因芯片检特异性高、敏感性强，可用于 三种病毒的快速检测与甄别。 关键词：克里米亚_阿0果出血热裂谷热拉沙热荧光基因芯片可视化 基因芯片 万方数据 THE THE ESTABLISHA们狲T 0F FLUORESCENTⅫCROARRAY AND VISUALIZED NⅡCRoARR AY FOR THREE TYPES OF VIRAL HEMORRHAGIC FEVERS AB STRACT Viral hemorrhagic fevers refer to a group of infectious diseases caused by four families of RNA viruses．All of these illnesses are characterized by fever and bleeding．At present，there is no effective vaccine and therapeutic drugs for most viral hemorrhagic fevers． Crimean-Congo hemorrhagic fever is a tick-borne viral disease caused by Congo Crimea hemorrhagic fever virus(CCHFV)．There are reports of infection cases from more than 3 0 countries in Africa，Asia，South-Europe，and the Middle East．Rift Valley fever is a zoonotic disease caused by Rift Valley fever virus(RVFV)．The disease is epidemic in the African，the Middle East and the Yemen area，can harm personal safety and the development of animal husbandry． The Lassa virus(LAV) causes Lassa fever，a viral hemorrhagic fever that is endemic in West Africa．Because the disease is highly contagious，some European and American countries have also appeared infection case． At present，techniques for the diagnosis viral hemorrhagic fever include III 万方数据 virus virus isolation,detection of specific antibody responses，and detection o