4种重要虫媒病的核酸液相芯片高通量检测方法的建立  

Development of Liquichip Technique to Detect Four Insect-borne Disease Virus Rapidly

作　　者：詹爱军[1] 王新卫[2] 卢体康[1] 陈书琨[1] 孙洁[1] 陈兵[1] 曾少灵[1] 花群义[1] 

机构地区：[1]深圳出入境检验检疫局动植物检验检疫技术中心,深圳518010 [2]河南农业大学牧医工程学院,郑州450002

出　　处：《畜牧兽医学报》2010年第2期240-245,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：科技部"863"计划项目(2006AA10Z445);国家质检总局项目(2008IK011)资助

摘　　要：为建立可检测鹿流行性出血热病毒（EHDV）、阿卡斑病毒（AKV）、蓝舌病病毒（BTV）和水泡性口炎病毒（VSV）的液相芯片快速检测技术,用DNAStar软件对GenBank中BTV的VP7基因、EHDV的VP7基因、AKV的N基因和VSV的NP基因序列进行序列分析,设计针对这些基因的特异性探针并标记生物素,分别与不同编号的荧光编码微球偶联后再与这些病毒相应基因的PCR产物杂交反应,用液相芯片检测仪（Liquichip 200）检测荧光信号建立了以上4种虫媒病的快速液相芯片检测方法。检测结果显示,该方法具有较好的特异性,偶联特异性探针的微球只与相应的病毒基因的PCR产物反应,而不与其他虫媒病病毒反应;检测灵敏度达到50~100个TCID50。本研究建立了可以同时检测鹿流行性出血热病毒、阿卡斑病毒、蓝舌病病毒和水泡性口炎病毒的快速高通量液相芯片技术,为其他类似病毒的快速高通量检测提供了借鉴和经验。The aim is to develop liquichip technique to detect infection of epizootic haemorrhagic disease virus of deer（EHDV）,akabane virus（AKV）,bluetongue virus（BTV）,and vesicular stomatitis virus（VSV）.The VP7 gene of EHDV,N gene of AKV,VP7 gene of BTV and NP gene of VSV in the GenBank were analyzed by using the software DNAStar 5.0.Specific gene probes of all these four viruses labeled with biotin were prepared and coupled with fluorescence-coded microspheres.The probes were used for hybridization reaction to RT-PCR products of the four viruses,and then the liquichip detection technique for detection of all these four viruses was established by using liquichip 200 to detect fluorescence signals in the reaction system.The results showed that this assay method displayed better specificity to RT-PCR products of correspondent viruses and no response to that of other insect-borne disease viruses when being used for detection.The sensitivity test indicated that the detecting limitation for the viruses could reach to 50-100 TCID50.The rapidly high throughput liquichip detection technique to detect the four insect-borne disease viruses was established,which provided a foundation and exploration for further research to detect other viruses with the same technique in animal husbandry.

关 键 词：鹿流行性出血热病毒 阿卡斑病毒 蓝舌病病毒 水泡性口炎病毒 液相芯片检测技术 

分 类 号：S854.43[农业科学—临床兽医学]