IGS1、ITS和D1／D2区在毛孢子菌鉴定中的意义及国内阿萨希毛孢子菌基因分型研究

Significance of IGS1, ITS and D1/D2 regions in molecular identification of Trichosporon species and genotype of T. asahii in China

目的比较不同DNA区域在毛孢子菌分子学鉴定方面的灵敏性和特异性以及国内临床分离5株阿萨希毛孢子菌（rasahii）基因型的研究。方法用玻璃珠法提取总DNA，用DI／D2（26S）、ITS、IGS1区的特异性引物进行PCR扩增，克隆、测序；用CLUSTAL X1．83进行比对分析，构建系统发生树并进行基因分型。结果T.asahii（CBS2479）、真皮毛孢子菌（T.dermatis）、赖巴克斯毛孢子菌（T.laibachii）3株菌D1／D2长度分别为：640、639、637bp；ITS区长度分别为：541、528、531bp；IGS1区长度分别为：643、515、411bp。临床分离5株rasahii IGS1区长度均为643bp。所有毛孢子菌D1／D2区序列相似性为：89％-99％，ITS区为85％-99％，IGS1区为11％～95％。临床分离菌株BZP07001，BZP07002，BZP07004，BZP07005属于基因型Ⅰ，菌株BZP07003属于基因型Ⅳ。结论在鉴别系统发生关系较近的毛孢子菌时IGS1区序列分析优于DI／D2、ITS区，IGS1区序列分析具有更高的灵敏性和特异性。国内临床分离5株zasahii分属基因型Ⅰ和Ⅳ。

Objective To compare the sensitivity and specificity in molecular identification in different DNA regions of Trichosporon species and to study the genotype of T. asahii isolated from clinical specimens in China. Methods DNA was extracted from the cells of all experimental strains by using a method of glass bead method. The D1/D2, ITS and IGS1 regions were amplified by PCR with specific primers, the PCR products were cloned and sequenced. The sequences were referred to GenBank and compared with the other sequences of the Trichosporon species from GenBank by the software CLUSTAL X 1.83. Phylogenetic trees were constructed and genotypes were determined. Results The D1/D2 regions in T. asahii（ CBS2479）, T. dermat/s, and T. laibachii were 640,639, and 637 bp in length respectively, the ITS regions were 541, 528, and 531 bp respectively in length, and the IGS1 regions were 643, 515, and 411 bp respectively. The sequence similarity of the D1/D2 region was 89% -99%, that of ITS and IGS1 regions were 85% - 99% and 11% - 95% respectively. The clinically isolated strains BZP07001, BZP07002, BZP07004, and BZP07005 belonged to genotype Ⅰ , and the strain BZP07003 to genotype Ⅳ. Conclusion The sensitivity and specificity of the IGS1 region was higher than those of the D1/D2 and ITS regions in identification of phylogenetically closely related Trichosporon species. T. asahii isolated from clinical specimens in China belongs to either genotype Ⅰ or genotype Ⅳ.