中华皮肤科杂志 ›› 2010, Vol. 43 ›› Issue (8): 523-525.

• 论著 •    下一篇

PCR-RFLP技术快速鉴定八种致病丝状病原真菌的实验研究

张晓利1,吕雪莲2,沈永年3,吕桂霞3,王淼淼1,葛一平4,刘维达3   

  1. 1. 中国医学科学院皮肤病研究所
    2. 大连市皮肤病医院
    3. 南京 中国医学科学院北京协和医学院皮肤病研究所
    4.
  • 收稿日期:2010-03-23 修回日期:2010-04-22 出版日期:2010-08-15 发布日期:2010-08-10
  • 通讯作者: 张晓利 E-mail:zxl415@hotmail.com
  • 基金资助:

    科技重大专项：传染病检测技术研究-传染病病原体诊断和组合检测技

Rapid identification of eight pathogenic filamentous fungi with PCR-RFLP analysis

  • Received:2010-03-23 Revised:2010-04-22 Online:2010-08-15 Published:2010-08-10

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摘要：

目的 建立能快速鉴定深部丝状真菌感染病原菌的PCR-RFLP方法。方法 用真菌通用引物扩增烟曲霉、黄曲霉、土曲霉、黑曲霉、杂色曲霉、构巢曲霉、尖端赛多孢和串珠镰刀菌的ITS区，分别用HhaⅠ、HaeⅢ、HinfⅠ、TaqⅠ和MspⅠ 5种限制性核酸内切酶对PCR产物进行酶切，建立以PCR为基础的RFLP方法，然后对22株临床株和2株环境分离株进行PCR-RFLP图谱分析。结果 对PCR产物进行RFLP分析可以准确鉴定8种深部致病丝状真菌，从DNA提取到酶切分析可以在1个工作日完成。22株临床株和2株环境分离株PCR-RFLP鉴定结果与传统的形态学鉴定结果一致。结论 PCR-RFLP技术是一种能够快速鉴定丝状真菌的有效方法。

关键词: 多态性，限制性片段长度, 方法, 真菌

Abstract:

Objective To develop a PCR-RFLP method to rapidly identify filamentous fungi causing deep infection. Methods Universal fungal primers were used to amplify the internal transcribed spacer （ITS） region of Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus niger, Aspergillus versicolor, Aspergillus nidulans, Scedosporium apiospermum and Fusarium moniliforme followed by restriction fragment length polymorphism （RFLP） analysis with restrictive endonucleases Hha I, Hae III, Hinf I, Taq I and Msp I. Then, 22 clinical and 2 environmental fungal isolates were identified with the developed PCR-RFLP method. Results The RFLP analysis of PCR products with restrictive endonucleases Hha I and Hinf I allowed discrimination of 8 filamentous fungi causing invasive infection, and it took only 1 day to carry out the whole procedure from DNA extraction to PCR and restriction digestion. The identification results of 22 clinical strains and 2 environmental isolates with this PCR-RFLP method were completely consistent with those with conventional morphological method. Conclusion PCR-RFLP analysis is an efficient method for rapid identification of cultured filamentous fungi.

Key words: Fungi, Polymorphism, restriction fragment length, Methods