用ITS和TUB序列差异鉴定临床相关波氏假阿利什霉／尖端赛多孢

Identification of PeudaUescheria boydii/Scedosporium apiospermum clinical isolates by sequence differences of ITS and TUB

目的 探讨用rDNA基因ITS及TUB的差异对15株致病性波氏假阿利什霉/尖端赛多孢重新分类鉴定,以了解致病菌株种类及指导临床治疗的意义.方法 对15株北京大学真菌与真菌病研究中心保存的临床相关波氏假阿利什霉/尖端赛多孢采用形态学方法观察菌落形态及显微镜下特征,糖同化试验方法观察菌株对D-核糖的利用,以及对菌株ITS及TUB进行Clustal X序列比对和MEGA 4方法种系发生研究.结果 15株波氏假阿利什霉/尖端赛多孢菌落形态学和显微镜特征无明显差异,仅1株出现有性期表现.糖同化试验显示所有菌株均可利用D-核糖.15株菌经与荷兰CBS网站（http：//www.cbs.knaw.nl）数据库中模式株比对以及绘制种系发生图,显示分属于波氏假阿利什霉复合种第4支Scedosporium apiospermum和第5支Pseudallescheria boydii,且分别为4和11株.结论 对于波氏假阿利什霉复合种仅依赖形态学和生理生化特征难以准确鉴定,且耗时长,而通过多位点基因片段序列分析能可靠鉴定波氏假阿利什霉/尖端赛多孢.对临床相关的波氏假阿利什霉/尖端赛多孢建议在形态学基础上结合分子方法进行种水平鉴定.

Objective To re-identify fifteen clinical Pseudallescheria boydii/Scedosporium apiospermum isolates by the sequence difference of ITS rDNA and partial β-tubulin gene （TUB） and thus understand the pathogenic strain types for guiding the clinical treatment.Methods Morphological appearances,D-ribose assimilation and sequencing of ITS and TUB were used to re-identify the fifteen clinical strains of Pseudallescheria boydii/Scedosporium apiospermum.The sequences of ITS and TUB were analyzed with Clustal X and MEGA 4 software.Results No difference of morphological appearances was found in the fifteen strains.Cleistothecium was observed in one isolate.All the strains were D-ribose assimilation positive.The clinical strains were re-identified as P.boydii species complex by the CBS database （http：//www.cbs.knaw.nl）. Eleven strains were P.boydii and four strains were S.apiospermum respectively.Conclusions P.boydii and S.apiospermum cannot be identified correcdy by the time-consuming conventional morphological method and biochemical characteristics.The study recommend that the clinical isolates of P.boydii and S.apiospermum should be identified utilizing a combination of traditional phenotype method and molecular biotechnology.