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  

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

作　　者：薛秀娟[1] 郑和平[1] 李国明[2] 黄进梅[1] 曾维英[1] 薛耀华[1] 吴兴中[1] 

机构地区：[1]广东省皮肤性病防治中心,广州510500 [2]广东医学院

出　　处：《中华皮肤科杂志》2009年第12期814-816,共3页Chinese Journal of Dermatology

基　　金：广东省科委资助项目（2008B0315）

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16SrRNA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1CFU。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＋Uu2例，Uu＋Mg1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。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 pareum （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.

关 键 词：支原体属 寡核苷酸探针 聚合酶链反应 

分 类 号：R450[医药卫生—治疗学]