三种出血热病毒的荧光基因芯片检测方法的建立  

Development of fluorescent gene chip for detection of three types of hemorrhagic fever virus

作　　者：曹佳媛 田明尧[2] 崔卓栋 柴丹 范园园[2] 赵飞[2] 张艳芳[2] 金宁一[2] 

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]军事医学科学院军事兽医研究所,吉林长春130122

出　　处：《中国兽医科学》2016年第1期1-7,共7页Chinese Veterinary Science

基　　金：国家科技支撑计划项目(2010BAD04B02);公益性行业(农业)科研专项(201203082)

摘　　要：为建立可同时检测新疆出血热病毒(CCHFV)、裂谷热病毒(RVFV)、拉沙病毒(LSV)的荧光基因芯片,分别针对CCHFV和RVFV的S基因、LSV的NP基因,应用生物学软件设计了3对引物与6条探针。运用多重PCR扩增和标记目的片段,将其与基因芯片杂交,最后扫描分析结果。分别对杂交温度与杂交时间进行条件优化,最后对基因芯片的特异性、敏感性和重复性进行评估。结果显示,设计的所有探针都可以与目的片段特异性杂交,探针1a与1b可检测到CCHFV,探针2a与2b可检测到RVFV,探针3a与3b可检测到LSV。相比多重PCR,荧光基因芯片检测CCHFV的灵敏度提高了10倍,检测RVFV的灵敏度提高了100倍,检测LSV的灵敏度提高了100倍。结果表明,本试验建立的同时检测3种出血热病毒的荧光基因芯片检测方法特异性高、敏感性强,可用于这3种病毒的快速检测与甄别。The aim of the present study was to establish a quick and sensitive method to simultaneously detect Crimean-Congo hemorrhagic fever virus（CCHFV）,Rift Valley fever virus（RVFV）and Lassa virus（LSV）using fluorescent gene chip.Then the detection method was optimized.Three pairs of primers and six oligonucleotide probes were designed with bioinformatics software in accordance with the conserved sequences of the CCHFV,RVFV S gene and LSV NP gene and oligonucleotide microarrays were prepared.Using multiple PCR amplified and labeled purpose fragments,the fragments were hybridized with probes on the gene chip,then the results were recorded.The hybrid time and hybrid temperature were optimized,then the use of fluorescent gene chip in terms of specificity,sensitivity and reproducibility was evaluated.The results showed that all probes were hybridized with correspondingly samples and had strong positive signals：probes 1aand 1bspecifically detected CCHFV,probes 2aand 2bspecifically detected RVFV,probes 3aand 3bspecifically detected LSV.Compared with multiple PCR,the sensitiveness of gene chip detecting CCHFV increased 10 times,the sensitiveness of gene chip detecting RVFV increased 100 times,and the sensitiveness of gene chip detecting LSV increased 100 times.In conclusion,this study has established a kind of fluorescent gene chip for detection of three kinds of hemorrhagic fever virus with high sensitivity and specificity.The method can be used for rapid detection of the three kinds of hemorrhagic fever virus.

关 键 词：新疆出血热病毒 裂谷热病毒 拉沙病毒 多重PCR 荧光基因芯片 

分 类 号：S852.659.5[农业科学—基础兽医学]