猪传染性胃肠炎病毒检测基因芯片的构建  

Construction of detecting genechip for transmissible gastroenteritis virus

作　　者：曹三杰[1] 黄小波[1] 文心田[1] 肖国生[1,2] 

机构地区：[1]四川农业大学动物医学院动物传染病与基因芯片实验室／动物疫病与人类健康四川省重点实验室,四川雅安625014 [2]重庆三峡学院生物系,重庆404000

出　　处：《中国兽医学报》2009年第2期121-124,共4页Chinese Journal of Veterinary Science

基　　金：教育部长江学者和创新团队发展计划资助项目(IRT0555-9)

摘　　要：采用PCR扩增制备TGEV的靶基因并进行纯化，对基因芯片的最佳靶基因点样质量浓度、探针质量浓度、杂交温度、杂交时间进行筛选，选择构建检测芯片的最适靶基因，进行基因芯片探针最佳标记方法试验。结果表明，质粒PCR扩增和采用异丙醇沉淀纯化的靶基因质量好，基因芯片最佳靶基因点样质量浓度为200mg／L，最佳探针质量浓度为3000μg／L，预杂交时间为1h，杂交时间为3～6h，杂交温度为48℃，以S、S3、sM、M、N、ORF7、POL等7个靶基因为构建TGEV检测芯片的最适靶基因，确定了多重PCR扩增标记TGEV探针的最佳体系，Cy3-dCTP标记浓度为2．5μmol／L，构建的TGEV检测芯片与标记的混合探针杂交效果好。A batches of target genes were amplified from recombinant plasmid of TGEV genes, the optimal target genes concentration, probe concentration,hybridization temperature and hybridization time were explored, the target genes for constructing TGEV genechip was chosen and the probes labeling method was tested. The results showed that the optimal target gene concentration was 200 mg/L, the optimal probe concentration was 3 000 μg/L,the prehybridization time was 1 h, the hybridization time was 3-6 h and the hybritization temperature was 48℃. The gene S, sM,M,N,ORF7 and POL were deterimined to be the target genes of TGEV genechip. Multiplex PCR was used to label the multiple TGEV probes, the hybridization results was good.

关 键 词：猪传染性胃肠炎病毒 检测 基因芯片 构建 

分 类 号：S852.15[农业科学—基础兽医学]