ITS结合RPS0两步PCR快速鉴定临床5种假丝酵母菌  

Two steps PCR of ITS combined with RPS0 genes for rapid identifying 5 species of Candida

作　　者：吴金燕[1] 张蕾蕾[2] 易国辉[1] 刘飞[1] 周利民[1] 王华民[3] 郭虹[1] 

机构地区：[1]海南医学院科学实验中心 [2]海南省人民医院急诊科 [3]海南医学院热带医学与检验学院,海南海口571101

出　　处：《海南医学院学报》2012年第3期292-296,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);国家自然科学基金资助项目(30760001)~~

摘　　要：目的:建立快速准确鉴定常见假丝酵母菌种类的基因检测技术。方法:提取假丝酵母菌基因组DNA;第一步PCR用通用引物扩增ITS基因;第二步三重PCR用种特异性引物扩增RPS0基因;分别对已知及未知样本检测、验证。结果:ITS基因扩增,光滑假丝酵母菌(Cg)和克柔假丝酵母菌(Ck)分别得到与预期结果吻合的881bp和419bp的PCR产物;白假丝酵母菌(Ca)、热带假丝酵母菌(Ct)和近平滑假丝酵母菌(Cp)的ITS扩增片段均略大于500bp;RPS0三重PCR对Ca、Ct和Cp DNA扩增,分别得到约310bp、147bp及110bp的片段,与预期结果相符;30株已知假丝酵母菌两步PCR检测结果,种类符合率为100%;43株未知样本两步PCR鉴定结果,经RPS0扩增产物序列测定,符合率亦为100%。结论:建立了ITS通用基因结合RPS0种特异基因鉴定5种常见假丝酵母菌种类的两步PCR技术,该技术简单、快速、准确及廉价;对双重感染患者更有鉴别优势,有临床应用价值。Objective. To devetop a rapid and accurate PCR method for identifying clinical common Candida. Method. Genomic DNA was extracted from Candida samples for two steps PCR assays. The first step PCR was amplifying fungal universal ribosome RNA internal transcription spacer （ITS） gene. The second multiplex PCR was amplifying three speciespecific ribosome proteins family （RPS0） gene. These two steps PCR were used for confirming known and identifying unknown species samples of Candida, respectively. Results. ITS gene PCR showed that 881bp and 419bp fragments, which indicated Candidaglabrata （Cg） and Candida kruseis （Ck）, respectively, in line with expectations. ITS gene fragments for Candia albicans （Ca）, Candia tropicalis （Ct）, and Candia parapsilosis （Cp） were all around about500bp. The result of RPS0 gene triple PCR displayed three razes ot 310bp, 141 bp anti liuDp, represenung for Ca, Ct and Cp, respectively, and were consistent with the expected results. For 30 identified strains, the results of two steps PCR showed 100M coincidence rate and for 43 clinical samples of Candida unknown species came out the same results with post verification of RPS0 PCR fragments sequencing. Oonclusions：Two steps PCR technique was developed by using ITS combined with RPS0 genes for rapid identi- fying 5 species of Candida. It has been proved to be a simple, rapid, accurate and specific technique, with more advantage to identify dual Candida infection and clinical application value.

关 键 词：假丝酵母菌 PCR鉴定 内部转录间隔区 核糖体蛋白家族 

分 类 号：R781.5[医药卫生—口腔医学] R379[医药卫生—临床医学]