18SrRNA基因序列分析在临床常见酵母样真菌鉴定中的应用

Identification for medically important yeast-like fungal species by sequence analysis of 18S rRNA gene

目的 应用18SrRNA基因序列分析技术对临床分离的常见酵母样真菌进行种的分类鉴定，且与传统方法比较，分析基因序列分析法鉴定临床常见酵母样真菌的可行性。方法收集北京同仁医院微生物室菌库酵母样真菌115株，提取的DNA用18SrRNA通用引物进行PCR扩增，扩增产物直接测序，测序结果提交GenBank通过核酸序列比对对微生物种属进行鉴定，同时进行真菌显色培养基鉴定、Vitek32YBC鉴定，比较3种不同方法鉴定酵母样真菌的种鉴定准确率，阐明应用基因序列分析法鉴定临床酵母样真菌的可行性。结果18SrRNA基因序列分析法与表型鉴定法相比较，有13株酵母样真菌鉴定结果存在差异：显色培养基仅鉴定出102株，与基因序列分析鉴定的符合率为89．2％（91／102），Vitek32YBC鉴定结果与基因序列分析鉴定的符合率为91．3％（105／115）；显色培养基、Vitek32YBC和18SrRNA基因序列分析鉴定到种的比例分别为88．7％（102／115）、100％（115／115）、100％（115／115）。结论18SrRNA基因序列分析法可对常见酵母样真菌进行准确分类。

Objective To compare sequence analysis of the yeast-like fungal isolates with traditional methods and analyze the feasibility of identification of common yeast-like fungal by sequence analysis of gene. Methods 115 yeast-like fungal isolates were collected in the clinical laboratory of Beijing Tongren Hospital. DNA of yeast-like fungal was extracted and then amplified with universal primers of part of 18S rRNA genes followed by sequencing directly. The sequences obtained were submitted to the GenBank （NCBI） to identify the fungi. At the same time, the CHROMagar Candida and Vitek 32 YBC were used to identify the fungi. The identification accuracy with three methods was compared to explore the feasibility of the identification of sequence analysis. Results 18S rRNA gene sequence analysis was compared with traditional method. There were some differences in the identification results of 13 strains. The coincidence rate between CHROMagar Candida and sequence analysis was 89. 2% （91/102）and the coincidence rate between Vitek 32 YBC and sequence analysis was 91.3% （ 105/115 ）. The positivity rate of species-level identification by CHROMagar Candida , Vitek 32 YBC and the 18S rRNA gene sequence analysis were 88.7% （ 102/115 ）, 100% （ 115/115 ）, 100% （ 115/115 ）. Conclusion Identification of medically important yeast-like fungal by sequence analysis of the 18S rRNA gene is reliability.