7种假丝酵母菌耐药相关基因GeXP检测方法的建立  

Establishment of GeXP technique for detecting 7 resistant marker genes of Canadida spp

作　　者：吴金燕[1] 易国辉[1] 周利民[1] 黄宪希[1] 薛丽[1] 薛伟玲[1] 郭虹[1] 

机构地区：[1]海南医学院科学实验中心,海南海口571199

出　　处：《海南医学院学报》2014年第11期1477-1481,共5页Journal of Hainan Medical University

基　　金：海南省重点科技资助项目(090214);海南医学院科研培育基金(2010-009);海南省教育厅基金(Hjkj2012-36)~~

摘　　要：目的:利用GeXP多重基因表达遗传分析系统,建立一种可同时检测7种假丝酵母菌耐药相关基因RNA表达水平的方法。方法:设计7种(MDR1、FlU1、CDR1、CDR2、ADH1、ERG3、ERG11)已知与白假丝酵母耐药相关基因的特异性引物及假丝酵母ITS基因参照引物,优化多重反应体系及反应条件,用热带假丝酵母标准株来验证多重反应体系的特异性,通过比较标准株和临床敏感株,剂量依赖株及耐药株各基因表达峰图,分析不同菌株各基因表达差异。结果:经过优化反应条件,可同时扩增出假丝酵母耐药相关的7种基因特异片段,耐药株和剂量依赖株的ERG11、CDR2、CDR1、MDR1和ERG3基因的相对表达量与敏感株相比均有不同程度的增高。结论:建立了多重基因表达检测体系,GeXP多重基因表达检测方法具有高通量、特异性强、灵敏度高和快速等优点。Objective： To establish a multiplex RT-PCR method for detecting 7 resistant marker genes expression simultaneously by using GeXP multi gene genetic analysis system. Method： Specific primers of 7 Candida albicans resistance marker genes, MDR1, FLU1, CDR1, CDR2, ADH1, ERG3, ERGll and internal control gene, ITS were designed by GeXP eXpress Profile software. RNAs were isolated from Candida tropicalis strains which were standard strain, strains with resistance, intermediate （dose dependent） and sensitive to FLuconazole using Yeast RNAiso Kit. The protocols for Gexp reactions were optimized and the results were analyzed by GeXP reaction software. Result.. RNA expression of all 7 genes could be detected in the same reaction system. The relative expression levels of ERG11, CDR2, CDR1, MDR1 and ERG3 from Fluconazole resistance and dose dependent strains were higher than those of sensitive strains. Conclusion： A GeXP based multiplex PCR technique for detecting 7 Candida albicans resistance marker genes has been developed with advantages of higher throughput, stronger specificity and higher sensitivity.

关 键 词：多重基因表达 假丝酵母菌 耐药基因 

分 类 号：R34[医药卫生—基础医学]