摘要
目的建立基于聚合酶链反应的单链构象多态性分析(PCR-SSCP)技术,对引起常见医院感染的光滑假丝酵母菌进行基因多态性分析,并探讨改善其灵敏度和分辨率的优化条件。方法采用PCR-SSCP技术对临床分离的19株光滑假丝酵母菌以及1株标准株ATCC90030的rDNA的ITSⅠ区(引物ITS1/ITS2)和ITSⅡ区(引物ITS3/ITS4)多态性进行分析,并对PCR-SSCP的条件进行优化。结果ITSⅠ区较ITSⅡ区保守性低,变异性较强,较为适合SSCP分型。针对ITSⅠ区,SSCP电泳在无甘油时,4℃和15℃均将菌株分为6型,但各型所含菌株不同;存在5%甘油时,SSCP电泳分辨率明显增高。结论在15℃及5%甘油存在的条件下,针对ITSI区的PCR-SSCP分型是一种快速、简便的适合于致医院感染光滑假丝酵母菌分型的新流行病学研究手段。
Objective To set up a new method of molecular epidemiology for Candida glabrata in nosocomial infection based on polymerase chain reaction and single-strand conformation polymorphism analysis（PCR-SSCP）, and investigate its sensitivity and resolving power. Methods Nineteen clinical strains of Candida glabrata were isolated. The ITS Ⅰ and ITS Ⅱ regions of these strains and one ATCC strain（ATCC 90030） were amplified using two pairs of prime, ITS1/ITS2 and ITS3/ITS4, respectively. The amplified products were analyzed by SSCP and the electrophoresis conditions were optimized. Results Compared to ITSⅡ region, ITS Ⅰ was less conservative and more variable, thus was more suitable for SSCP analysis. The 19 clinical strains were classified into 6 types by ITSⅠ region at either 4℃ or 15℃ in the absence of glycerol, however, the composition of each type were different. The addition of 5% glycerol in the electrophoresis significantly improved the resolving power of PCR-SSCP. Conclusion At 15℃ in the presence of 5% glycerol, PCR SSCP targeting on ITS Ⅰ region might be a quick, simple and reliable method for molecular epidemiological study on nosocomial infection caused by Candida glabrata.
出处
《中国感染控制杂志》
CAS
2008年第3期152-156,共5页
Chinese Journal of Infection Control
关键词
光滑假丝酵母菌
PCR-SSCP
流行病学研究
真菌
Candida glabrata
polymerase chain reaction and single-strand conformation polymorphism analysis （PCR-SSCP）
epidemiological study
fungus
