ITS基因测序分析对89株病原真菌鉴定的应用评价

Application and evaluation for sequencing analysis based on internal transcribed spacer gene for identification of 89 strains of pathogenic fungi

目的探讨内转录间隔区(ITS)基因扩增测序技术在病原真菌鉴定中应用价值。方法应用通用引物ITS1和ITS4对收集的89株真菌(包括酵母菌标准菌株6株,室间质评菌7株,临床分离的酵母样真菌42株和丝状真菌34株)进行PCR扩增和基因测序。测序成功的ITS序列在CBS数据库(http://www.cbs.knaw.nl/Collections)进行序列比对,结合Mycobank(http://cn.mycobank.org/)分类信息以获得其菌种鉴定信息。结果 89株不同种类的真菌均成功扩增到ITS靶基因,经测序,89株真菌均获得扩增的全序列。序列比对结果显示,6株酵母菌标准株中,5株鉴定到种,1株鉴定到属;7株室间质评株中,1株酵母菌鉴定到种,6株丝状真菌中2株鉴定到种,1株鉴定到属,3株鉴定到复合群;42株临床分离的酵母样真菌中,39株鉴定到种,3株鉴定到属;34株临床分离的丝状真菌中,10株鉴定到种,18株鉴定到属,6株鉴定为复合群。结论通用引物ITS1和ITS4广谱性强。ITS基因序列扩增和测序分析可快速、有效鉴定临床疑难真菌,但仍存在数据分析过程烦琐和结果解释困难的问题。

Objective To explore the application of internal transcribed spacer（ITS） gene sequencing in identification of pathogenic fungi. Methods PCR amplification and gene sequencing were performed by the universal primer ITS1 and ITS4 of fungal ITS gent for 89 strains of fungi, including 6 standard strains of yeasts, 7 strains of external quality assessment, 42 strains of clinically isolated yeast- like fungi and 34 strains of filamentous fungi. The amplified sequences were analyzed and compared with those of CBS database （ ht- tp ：//www. cbs. knaw. hi/Collections） and the taxonomic information providing by Mycobank （http ：//cn. mycobank, org/） to obtain the information of species identification. Results All of the 89 strains of fungi were successfully amplified into ITS target gene and the re- sults of sequencing demonstrated the entire sequences of 89 strains were obtained. The sequence alignment showed that 5 of 6 standard strains of the yeasts were identified to species and 1 strain was identified to genus. Among 7 strains of external quality assessment, 1 strain of yeast was identified to species, 2 strains of 6 filamentous fungi were identified to species, 1 strain was identified to genus and 3 strains were identified to complex group. Among 42 strains of chnically isolated yeast-like fungi, 39 were identified to species and 3 were identified to genus. Among 34 clinically isolated strains of filamentous fungi, 10 were identified to species, 18 were identified to genus and 6 were identified to complex group. Conclusion The universal primer ITS1 and ITS4 showed broad spectrum for amplifica- tion. The identification based on amplification and sequencing analysis of ITS gene could fast, effectively identify clinical suspicious fungus, but the process of data analysis and the result interpretation should still be difficult.