中华皮肤科杂志 ›› 2011, Vol. 44 ›› Issue (8): 556-559.

• 论著 • 上一篇    下一篇

菌落PCR技术快速鉴别丝状真菌的实验研究

张晓利1,吕雪莲2,沈永年3,吕桂霞3,王淼淼4,葛一平5,刘维达3   

  1. 1. 暨南大学临床医学博士后流动站
    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. 中国医学科学院皮肤病研究所
    5.
  • 收稿日期:2011-02-18 修回日期:2011-04-08 出版日期:2011-08-15 发布日期:2011-07-26
  • 通讯作者: 刘维达 E-mail:liumyco@hotmail.com
  • 基金资助:

    科技部重大专项;真菌感染新型诊断方法的建立及应用

Rapid identification of filamentous fungi by colony PCR

  • Received:2011-02-18 Revised:2011-04-08 Online:2011-08-15 Published:2011-07-26

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摘要：

目的 探讨菌落PCR在检测病原性丝状真菌方面的应用价值。方法 初步建立用于丝状真菌的菌落PCR检测技术，用19种丝状真菌标准株进行验证，所有菌落PCR扩增产物进行测序，并选取8种菌株的菌落PCR产物和酶切结果与常规PCR进行比较，检测其准确性和可靠性。结果 19株菌中有16株（84.2%）菌落PCR成功扩增内转录间隔（ITS）区，ITS区基因序列分析鉴定菌种正确，与NCBI数据库中相同菌种的相似度为96% ~ 100%；与常规PCR进行比较的8株菌中，除构巢曲霉菌落PCR扩增结果为阴性外，其他菌种菌落PCR产物及酶切条带与常规PCR基本一致。结论 与常规PCR相比，菌落PCR检测丝状真菌操作简单，省时省力，鉴定菌种具有较高的准确性和可靠性，可以用于丝状真菌的快速鉴定。

关键词: 方法

Abstract:

Objective To estimate the application value of colony PCR in the detection of pathogenic filamentous fungi. Methods Colony PCR was established and performed to amplify the internal transcribed spacer （ITS） region of 19 species （strains） of filamentous fungus followed by sequencing analysis. At the same time, DNA extracts from 8 of the 19 species of filamentous fungus were subjected to conventional PCR. Hha I and Hinf I endonucleases were used for restriction fragment length polymorphism （RFLP） analysis of the conventional and colony PCR products. Comparison analysis was carried out between the colony and conventional PCR. Results Of the 19 strains, 16（84.2%） yielded positive results by colony PCR; sequence analysis of the PCR products of ITS region revealed a 96% - 100% similarity with the reference sequence （NCBI database） of corresponding fungi. The amplification product length and RFLP profile of these products from the 8 species of filamentous fungus, except for those from Aspergillus nidulans, were consistent between the colony and conventional PCR. Conclusions Compared with conventional PCR, colony PCR-based detection of filamentous fungi is easy to operate, time and labor-saving, with high accuracy and reliability, and can be applied to the rapid identification of filamentous fungi.

Key words: Methods