中华皮肤科杂志 ›› 2008, Vol. 41 ›› Issue (8): 512-514.

• 论著 • 上一篇    下一篇

临床分离阿萨希毛孢子菌IGS1区克隆及RFLP分析

夏志宽 杨蓉娅 王文岭 敖俊红 王聪敏 张洁   

  1. 北京军区总医院皮肤科 北京军区总医院皮肤科 北京军区总医院皮肤科 北京军区总医院皮肤科
  • 收稿日期:2008-03-07 修回日期:2008-04-02 发布日期:2008-08-15
  • 通讯作者: 夏志宽 E-mail:sum7@sina.com

Cloning and restricted fragment length polymorphism analysis of intergenic spacer region 1 of clinical isolates of Trichosporon asahii

  

  • Received:2008-03-07 Revised:2008-04-02 Published:2008-08-15

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摘要： 目的 明确临床分离的5株阿萨希毛孢子菌（T. asahii）基因间隔区（IGS1）的序列，分析T. asahii种内基因多态性。方法 用玻璃珠法分别提取临床分离5株T. asahi及标准对照株总DNA，用ITS及IGS1区特异引物，PCR扩增目的区域，IGS1区扩增产物纯化后进行克隆、测序，测序结果分别提交到基因库，获取登录号。用BLAST和CLUSTAL X 1.83软件对序列进行比对分析。分别用限制性内切酶HapⅡ、MboⅠ、AluⅠ、HhaⅠ对ITS及IGS1区PCR产物进行酶切及RFLP分析。结果 6株菌均成功扩增出长度约为540 bp的ITS区及643 bp的IGS1区基因片段，IGS1区序列相似性为97% ～ 99%。RFLP分析发现，菌株BZP07003 IGS1区HapⅡ酶切结果及菌株BZP07004 IGS1区 HhaⅠ酶切结果与其他菌株不同，MboⅠ及AluⅠ酶切6株菌结果无差异，ITS区6株菌酶切结果无差异。结论 T. asahii IGS1区种内存在差异。HhaⅠ及HapⅡ可用于T. asahii种内多态性的分析研究，临床分离5株菌存在3种酶切结果，初步提示T. asahii临床分离株可能具有种内多态性及遗传差异。

关键词: 阿萨希毛孢子菌, IGS1, 克隆, RFLP

Abstract: Objective To sequence the intergenic sequence region 1 （IGS1） of 5 clinical isolates of Trichosporon asahii, and to analyze intraspecies polymorphism of IGS1 gene in T. asahii isolates. Methods Nuclear DNA was extracted from the 5 isolates of T. asahii by using a method of glass pearls. IGS1and internal transcribed spacer region （ITS） were amplified using specific primers by PCR. The products of IGS1 were cloned and sequenced using the ABI377 nucleotide sequenator. The resulted sequence of IGS1 was blasted against Genbank and aligned with CLUSTAL X 1.83 software. The restricted fragment length polymorphism （RFLP） analysis of IGS1 was performed using restricted endonucleases HapⅡ, MboⅠ, HhaⅠand AluⅠ. Results PCR yielded amplicons of ITS and IGS1 genes at the expected sizes of 540 bp and 643 bp, respectively, from all T. asahii isolates, with the sequence homology of IGS1 being 98% - 99% among the isolates. All isolates shared common RFLP profile after digestion of IGS1 amplicons with endonuclease MboⅠor AluⅠ, whereas the HapⅡ digestion profile of strain BZP07003 and HhaⅠdigestion profile of strain BZP07004 were distinct from that of other isolates. Conclusions HhaⅠand HapⅡ can be used to analyse the intraspecies polymorphism of T. asahii. Three RFLP profiles were achieved from these 5 T. asahii isolates by digestion with HhaⅠ and HapⅡ, suggesting that there is an intraspecies polymorphism and genetic heterogeneity within clinical isolates of T. asahii.

Key words: Trichosporon asahii, IGS1, Clone, RFLP