中华皮肤科杂志 ›› 2012, Vol. 45 ›› Issue (8): 545-548.

• 论著 • 上一篇    下一篇

随机扩增多态性DNA结合微流芯片鉴定马拉色菌的初步研究

王韵茹1,章强强2   

  1. 1. 上海复旦大学附属华山医院
    2. 上海复旦大学华山医院皮肤科
  • 收稿日期:2011-12-30 修回日期:2012-02-14 出版日期:2012-08-15 发布日期:2012-08-01
  • 通讯作者: 章强强 E-mail:zhangqq8@163.com

Random amplified polymorphic DNA combined with microfluidic chips in the identification of Malassezia species

Yun-Ru WANG1,Qiangqiang Zhang   

  • Received:2011-12-30 Revised:2012-02-14 Online:2012-08-15 Published:2012-08-01
  • Contact: Qiangqiang Zhang E-mail:zhangqq8@163.com

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摘要：

目的 探讨微流芯片在马拉色菌鉴定与分型中的应用优势。方法 收集马拉色菌标准菌株及直接镜检阳性的花斑糠疹患者皮损处皮屑及马拉色菌毛囊炎患者皮损处毛囊内容物培养出的马拉色菌菌株进行DNA测序，鉴定菌种，随机扩增多态性DNA聚合酶链反应（RAPD-PCR）电泳分析及微流芯片基因型定量分析并聚类分析树状图。结果 共分离83株马拉色菌临床菌株，其中72株分离自花斑糠疹，11株分离自马拉色菌毛囊炎。大多数菌株均可被2种随机引物（S22、S24）扩增而获得清晰条带，但以S22引物扩增的条带更为稳定、清晰，作为主要引物。不同种马拉色菌通过微流芯片基因型定量分析得到不同大小的固定阳性条带，所有菌株均可见种间和种内多态性。在DNA测序的基础上，使用RAPD结合微流芯片方法，基本可将8种马拉色菌（糠秕、合轴、球形、厚皮、斯洛菲、日本、大和及皮肤马拉色菌）区别。结论 RAPD结合微流芯片方法作为一种快速、高通量、高灵敏性的分析技术，在马拉色菌种间菌株遗传多样性、亲缘关系的分析及新种鉴定中显示出一定优越性。

关键词: 马拉色菌毛囊炎

Abstract:

Objective To evaluate the performance of microfluidic chips in the identification and genotyping of Malassezia species. Methods This study included 6 reference Malassezia strains and clinical Malassezia isolates from the scrapings of patients with pityriasis versicolor and follicular contents of patients with Malassezia folliculitis. These isolates were identified by DNA sequencing, random amplified polymorphic DNA （RAPD）-PCR and microfluidic chips. Cluster analysis was carried out and tree diagrams were generated. Results A total of 83 Malassezia isolates were obtained from 72 patients with pityriasis versicolor and 11 patients with Malassezia folliculitis. Genomic DNA of most strains was successfully amplified by PCR with two primers S22 and S24, and PCR with S22 primer produced more stable and clear amplification bands than that with S24. Positive bands of different sizes were repetitively obtained by using microfluidic chips, with interspecies and intraspecies polymorphisms observed in all the strains. On the basis of DNA sequencing, microfluidic chips and RAPD-PCR could be used to successfully distinguish the following eight species, i.e., M. furfur, M. sympodialis, M. globosa, M. pachydermatis, M. slooffiae, M. Japonica, M. yamatoensis and M. dermatis． Conclusions As a rapid, high-throughput and high-sensitivity method, microfluidic chips combined with RAPD-PCR shows an advantage for analyzing interspecies genetic diversity, genetic relationship of Malassezia species, as well as for identifying new Malassezia species.

Key words: Malassezia folliculitis