基因芯片技术检测重要人兽共患病病毒方法的建立  

Microarray for Screening and Identification of Important Zoonotic Viruses

作　　者：李永强[1] 康晓平[1] 孙庆歌[2] 刘洪[1] 常国辉[1] 祝庆余[1] 杨银辉[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,哈尔滨150001

出　　处：《中国生物化学与分子生物学报》2009年第11期1058-1063,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家高技术研究发展计划(863计划)资助(No.2007AA02Z406)~~

摘　　要：为了建立能对25种重要人兽共患病病毒进行筛查及鉴定用的基因芯片技术,本实验首先设计针对每种病毒的寡核苷酸探针并进行探针特异性的生物信息学验证.然后探索病毒核酸随机扩增方法,优化杂交动力学条件,建立本芯片标准的数据处理分析方法.最后用细胞培养的病毒和模拟临床标本验证芯片的敏感性与特异性.结果表明,锚定随机PCR扩增法适合于本芯片病毒核酸的扩增;芯片杂交前用0.25%NaBH4进行封闭,最优杂交条件为51℃,2h及50%甲酰胺浓度;芯片具有较好的敏感性及检测特异性.初步结果表明,本实验所建立的基因芯片技术可应用于对25种重要人兽共患病病毒进行筛查及鉴定.To screen and identify 25 zoonotic viruses, 300 viral oligonucleotide microarray probes were designed and validated by bioinformatic analyses. Random amplification of viral sequences were explored and the microarray hybridization conditions were optimized. The specificity and sensitivity of the customed microarray were evaluated with cell-cultivated viruses and simulated clinical samples. Before hybridization, the microarray was blocked with 0.25 % NaBH4 . 51 %, 2 hour incubation and 50 % formamide were applied for the optimal hybridization. All the 25 important zoonotic viruses were successfully identified by our screening in given samples. The results indicated that the anchored random PCR amplification of viral oligonucleotide was suitable for the microarray assays to detect zoonotic viruses.

关 键 词：基因芯片技术 人兽共患病病毒 筛查及鉴定 特异性 随机PCR 

分 类 号：Q789[生物学—分子生物学]