中华皮肤科杂志 ›› 2009, Vol. 42 ›› Issue (12): 814-816.

• 论著 • 上一篇    下一篇

反向斑点杂交技术快速鉴定泌尿生殖道致病性支原体的研究

薛秀娟1,郑和平2,李国明3,黄进梅4,曾维英4,薛耀华5,吴兴中6   

  1. 1. 河南省疾病预防控制中心性病艾滋病研究所
    2. 广州市广东省皮肤性病防治中心
    3. 湛江广东医学院微生物学免疫学教研室
    4. 广州市广东省皮肤病防治中心
    5. 广州市广东省皮肤病防治研究所
    6. 广东省皮肤性病防治中心
  • 收稿日期:2008-12-17 修回日期:2009-06-03 出版日期:2009-12-15 发布日期:2012-03-27
  • 通讯作者: 薛秀娟 E-mail:boboo2009@126.com
  • 基金资助:

    广东省科委资助项目（2008B0315）

Rapid detection of pathogenic mycoplasma in genitourinary tract using PCR-reverse dot blot hybridization

  • Received:2008-12-17 Revised:2009-06-03 Online:2009-12-15 Published:2012-03-27

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摘要：

目的 建立PCR反向斑点杂交方法（PCR-RDB）快速鉴定泌尿生殖道常见致病性支原体。方法 以4种支原体［微小脲原体（Up）、解脲脲原体（Uu）、生殖支原体（Mg）、人型支原体（Mh）］的16S rRNA基因为靶序列设计通用引物和探针，将4种特异的寡核苷酸探针固定于尼龙膜上。利用巢式PCR扩增Up、Uu、Mg和Mh，扩增产物变性后与各特异性探针进行杂交、显色并分析结果，并对检测体系的敏感性、特异性进行测试。同时检测分析60例临床拭子标本。结果 4种特异性探针只与相应的支原体DNA杂交，与其他病原体无交叉反应，其敏感性是1 CFU。60例临床拭子标本PCR-RDB方法共筛选出支原体阳性19例，其中3例为支原体混合感染的标本（Up + Uu 2例，Uu + Mg 1例）。结论 该方法可快速、敏感、准确地鉴定引起泌尿生殖道感染的致病性支原体。

关键词: 聚合酶链反应

Abstract:

Objective To develop a PCR-reverse dot blot hybridization （RDB） assay to rapidly detect pathogenic mycoplasmas in genitourinary tract. Methods Universal primers were designed and applied to amplify the 16S rRNA gene of ureaplasma parvum （Up）, ureaplasma urealyticum （Uu）, Mycoplasma genitalium （Mg）, Mycoplasma hominis （Mh） by using nested PCR. Specific nucleotide probes of Up, Uu, Mg and Mh were constructed and immobilized onto nylon membranes. PCR products were denatured and hybridized with specific oligonucleotide probes on nylon membrane. The sensitivity and specificity of the PCR-RDB assay were evaluated based on the hybridization results. Also, PCR-RDB was utilized to detect pathogenic mycoplasmas from 60 clinical samples. Results The four probes selectively hybridized with the PCR product of corresponding mycoplasmas, and no cross hybridization was observed. The detection limit of PCR-RDB was one colony forming unit （CFU） of mycoplasma. Out of the 60 clinical samples, 19 were positive for mycoplasm. Mixed infections were found in three samples, including two coinfected with Up and Uu and one with Uu and Mg. Conclusion PCR-RDB is a rapid, specific and sensitive approach to the identification of pathogenic mycoplasmas in urogenital tract.

Key words: PCR