目视化常见角膜致病真菌基因芯片的构建  

Visual gene chips for common pathogenic fungus in cornea

作　　者：张英朗[1] 王丽娅[2] 孙声桃[2] 李智涛 

机构地区：[1]郑州大学第一临床学院 [2]河南省眼科研究所 [3]河南省生物工程中心

出　　处：《山东医药》2007年第12期3-4,共2页Shandong Medical Journal

基　　金：河南省杰出人才创新基金项目(0321002000);河南省科技攻关计划项目(0623033000)

摘　　要：目的制备目视化常见角膜致病真菌基因芯片。方法将6属12种角膜致病真菌设计合成特异性寡核苷酸探针加尾后，固定于带正电荷尼龙膜的相应区域，用生物素标记的通用引物对12株标准菌株和82株临床分离株进行PCR扩增，将扩增产物与固定有寡核苷酸探针的尼龙膜进行反向斑点杂交，观测相应区域的斑点显色情况。结果12种真菌均产生了530～630 bp的PCR扩增产物，得到了具有各自特征的杂交后生物素-亲和素斑点显色图谱，可直接从该图谱上判断不同菌种。对82株临床分离株的检测敏感率为84．1％，未出现非特异性交叉反应。结论利用PCR结合反向斑点杂交技术制备目视化基因芯片，能够在3～4h完成对我国临床上常见的12种角膜致病真菌的菌种鉴定，特异性及敏感性较高。[Objective] To establish visual gene chips for common pathogenic fungus in cornea. [Methods] 12 standard strains and 82 clinical isolates of common pathogenic fungi in cornea were designed for species-specific oligonucleotide probes, and added homopolymer tails and then inoculated on a nylon membrane with positive charges. Fungi DNA was amplified by PCR with fungi-universal primers labeled with biotin. The PCR products were identified by hybridization with the species- specific probe fixed on the nylon membrane. [Results] All 12 species of fungi were amplified by PCR,and the length of PCR products was 530-630bp. 12 species-specific probes only hybridized with its target molecules,and the test manifested these probes were high specific. The results of testing 12 standard strains agreed with those of culture for all,the positive rate of testing 82 culture isolates is 87.8 %. [Conclusion] Using PCR with reverse dot blot hybridization to prepare visual gene chips can identify the common pathogenic fungi in cornea sensitively in 3-4 hours,it has a high specificity and sensitivity.

关 键 词：基因芯片 聚合酶链反应技术 反向斑点杂交技术 角膜病 真菌 

分 类 号：R772.21[医药卫生—眼科]