利用基因芯片快速检测多种临床常见致病菌及其耐药性基因  

Rapid detection of clinical pathogenic bacteria and antimicrobial-resistant genes using DNA chip

作　　者：毛红菊[1] 林东昉[2] 徐晓刚[2] 张宏莲[1] 张婴元[2] 汪复[2] 赵建龙[1] 

机构地区：[1]中国科学院上海微系统与信息技术研究所传感技术国家重点联合实验室,上海200050 [2]复旦大学附属华山医院抗生素研究所

出　　处：《中华传染病杂志》2006年第6期372-377,共6页Chinese Journal of Infectious Diseases

基　　金：中国科学院重大项目(KSCX1-06);卫生部部属(管)医疗机构临床学科重点项目(20011522)

摘　　要：目的快速、准确、灵敏地检测临床常见的致病菌及其耐药基因。方法采用基因芯片技术，利用细菌23S rRNA基因序列信息和某些耐药基因作为检测靶基因，设计针对不同菌属的寡核苷酸探针和耐药基因探针的基因芯片，聚合酶链反应（PCR）扩增并荧光标记目的DNA片段，通过杂交反应检测致病菌及其耐药基因。结果在理想状态下（检测试剂盒抽提的细菌基因组DNA）检测的结果与国内外文献报道的灵敏度相当（10^3～10^6细菌／ml），并在部分临床分离株、耐药标准菌株中进行验证，显示该方法有良好的特异性及可重复性。细菌种类能鉴别到种水平的有16种，能鉴别到属水平的有7类。覆盖了临床常见的多种病原菌，并且还可同期检测超广谱β内酰胺酶（ESBLs）基因耐药。结论DNA芯片检测具有快速、高特异性和高灵敏度的特点，是常规鉴定方法的一种有益补充。Objective To detect the clinical pathogenic bacteria and antimicrobial-resistant genes quickly and sensitively using DNA chip. Methods Based on the analysis of 23S rRNA gene sequences and other genes sequences associated with antimicrobial resistance （SHV〈CTX_ M）, oligonucleotide microarray was designed according to different bacteria and antimicrobial-resistant genes. The DNA fragments were amplified by labeling Cy5 fluorescence and detect clinical pathogenic bacterias and antimicrobial-resistant genes by hybridization. Results The result of detection （10^3-10^6 bacteria/ml） was consistent with that of some documents in domestic and overseas under ideal circumstances of detecting bacteria genomic DNA by the Reagent Box. And it was specific and reproducible when the detection system were evaluated with some clinical isolates and drug-resistant standard strain. DNA chip could identify 16 species and 7 generics including common diverse clinical pathogenic bacteria, and could detect the drug-resistant of extended spectrum β lactamase gene simultaneously. Conclusions The methods that we have established DNA chip is a sensitive, specific and reproducahle tool for supplying routine methods.

关 键 词：寡核苷酸序列分析 抗药性 微生物 基因 细菌 

分 类 号：R450[医药卫生—治疗学]