26S rDNA单链构象多态性分析在临床酵母菌菌种鉴定中的应用

摘要： 【目的】探讨酵母菌临床分离株26S rDNA D1/D2区序列种内相似性和种间差异性的快速检测方法，为临床酵母菌菌种鉴定方法的改进奠定基础。调查北京地区临床酵母菌的种群多样性，为国内酵母菌感染的流行病学研究提供新的基础数据。【方法】用5种常见临床酵母菌种的模式和权威菌株作为标准参考菌株，从北京四家综合性医院收集临床酵母菌260余株，PCR扩增其26S rDNA D1/D2区，对扩增产物进行单链构象多态性（Single-Strand Conformation Polymorphism，SSCP）分析和序列测定分析。【结果】常见病原酵母菌26S rDNA D1/D2区的SSCP图谱具有明显的种间差异性和种内相似性，可以通过该方法对菌株进行初步的菌种鉴定。D1/D2-SSCP和序列分析相结合，对260余株临床酵母菌进行了菌种鉴定，共鉴定有10个属20个种，优势属为念珠菌属(Candida)，优势种及其所占比例分别是：C. albicans (57.7%), C. parapsilosis (10.0%), C. tropicalis (9.2%), C. glabrata (6.7%)和C. krusei (5.8%)，并发现过去从未或很少报道致病的酵母菌种，愈来愈多地出现在临床分离菌株中。【结论】 26S rDNA D1/D2区的SSCP图谱分析为临床酵母菌株的快速鉴定提供了新的方法；北京地区酵母菌临床分离株呈种群多样性分布，C. albicans虽然仍占优势，但其它念珠菌种的比例已达42%。

Rapid identification of clinical yeast species by single-strand conformation polymorphism analysis of 26S rDNA D1/D2 domain

Abstract: Objective] To investigate the reliability of single-strand conformation polymorphism (SSCP) analysis of 26S rDNA D1/D2 domain for rapid identification of clinical yeast species and to examine the distribution of the yeast species in clinical strains from Beijing. Methods] Type and authentic strains of five common pathogenic yeast species were used as references. Approximately 260 yeast strains with diversified clinical origins were collected from four hospitals located in Beijing. The 26S rDNA D1/D2 domain of each strain was amplified by PCR and subjected to SSCP or sequence analysis. Results] SSCP analysis showed that the Candida strains with slight sequence differences in the D1/D2 domain could be effectively detected. The common pathogenic Candida species, including C. albicans, C. parapsilosis, C. tropicalis, C. glabrata and C. krusei, were clearly distinguished from each other by their SSCP patterns of PCR amplified D1/D2 domain products. Twenty species belonging to 10 genera were identified from the approximately 260 clinical yeast strains based on SSCP pattern comparison for the common species and D1/D2 sequence analysis for the uncommon species. The dominant species and their frequencies were: C. albicans (57.7%), C. parapsilosis (10.0%), C. tropicalis (9.2%), C. glabrata (6.7%) and C. krusei (5.8%). Conclusion] The results indicated that PCR-SSCP analysis of D1/D2 is a powerful approach for rapid species identification of clinical yeast strains. The most common clinical yeast species was C. albicans in Beijing but the increasing trend of non-albicans Candida species was observed.