ITS基因测序对须癣毛癣菌的鉴定分析

Identification of Trichophyton mentagrophytes by ITS gene sequencing

目的探讨ITS基因测序对须癣毛癣菌进行分子生物学鉴定的可行性,以确定致病菌株的种属。方法从病人头皮及头发分离出一株真菌,采用直接涂片镜检和培养的方法,观察其镜下形态和菌落特征,同时取纯培养物进行普通PCR,PCR产物经1%琼脂糖凝胶电泳分析,Tanon-3500凝胶成像系统进行鉴定,收集目的片段进行ITS基因测序。结果本菌的镜下形态和培养特征与其他毛癣菌属极其相似,难以直接鉴定分离菌为须癣毛癣菌,将其纯培养后用真菌通用引物ITS4、ITS5进行PCR扩增,所得的扩增片段大小约700 bp,将目的片段送至公司进行双向测序,测序结果经Blast比对,须癣毛癣菌与之相符率达99.5%。结论 ITS基因测序可以准确鉴定从临床分离的须癣毛癣菌。

Objective To investigate the feasibility of molecular biological identification of Trichophyton mentagrophytesby ITS gene sequencing so as to determine the species of pathogenic isolates,and provide direct evidence for clinical treatment.Methods A fungal strain was isolated from the patient＇s scalp and hair. Morphologic and colony characteristics of the isolatewere observed by direct microscopy and culture. Conventional PCR was performed on cultured fungal strain,PCR product waselectrophoresed in 1% agarose gel,and gel image was captured by Tanon-3500 gel imaging system. Target PCR fragment wascollected and sequenced by ITS gene sequencing. Results Cultural characteristics and microscopic morphology could notidentify the isolate as Trichophyton mentagrophytes. The size of PCR product was 700 bp,and the sequence of the isolatedisplayed 99.5% identity with Trichophyton mentagrophytes by Blast analysis. Conclusion ITS gene sequencing canaccurately identify the clinical isolates of Trichophyton mentagrophytes.