PCR-RFLP分析16s-23srDNA间区序列鉴定分枝杆菌菌种.pdfVIP

PCR—RFLP分析 16s~23srDNA间区序列鉴定分枝杆菌菌种 彭德虎 尹小毛 陈建波 广州市胸科医院 广东广州 510095 摘 要 目的 探讨16s～23srDNA间区序列DNAPCR扩增和限制性酶切分析在分枝杆菌分类鉴定中的价 值。方法 对19种分枝杆菌标准株的 16s～23srDNA间区序列DNA进行PCR扩增并对扩增产物进行限制 性内切酶naeIII、MSP1消化反应，分析不同菌种扩增片段及其限制性片段长度多态性的差异。结果，PCR 扩增结果显示：分枝杆菌一般扩增出1～2条带，缓慢生长分枝杆菌扩增片段在 340～480bP，快速生长分 枝杆菌扩增片段集中在470～575 bP，单从扩增产物的琼脂糖凝胶电泳只能鉴定33．3％的受试菌种，酶 切结果显示：分枝杆菌的酶切图谱彼此不同 结论 16s～23srDNA间区序列DNA PCR扩增和RFLP分析 是分枝杆菌分类鉴定的一种快速 、有效的方法。 关键词 分枝杆菌 PCR RFLP The vaIMe of mycobacteriaI strains identified by the identifica— tion of 16s~ 23s rDNA spacer region with PCR—RFLP PENG De——hu YIN Xiao——mao CHEN Jian、—bo Guangzhou city chesthospital， Guangzhou 510095 Guangdong China Abstract Objective To studythe value in the mycobacterialstrainsidentifiedbythe identification of 16s～ 23s rDNA spacer region with PCR-RFLP． Methods The 16s～ 23s rDNA spacer regions in 19 species of frequent mycobacterial standard strainswere amplified byPCR and theamplifiedproductsweresubjectedto RFLPusingtherestriction enzymes，Hae III， MspI， by which the differences among the amplified fragments and among the restriction fragment 1engthpolymorphism (RFLP)wereanalyzed．Results TheresultsofPCRamplifica— tion showed that1or 2 bands were always amp1ified in the mycobacterial strains，that the amplified fragment of slow grower mycobacteria was one between 340 and 480 bp， rapid grower mycobacteria 470 and 575 bp．33．3％ strains could be identified by the amplified products of agarosegelelectrophoresis(AGE)andtheresultsdigestedbytherestrictionenzymesshowed that the macrorestriction maps of the mycobacterial strains were different．Conelusions PCR amplification of the 16s～23s rDNA spacer regions and RFLP analysis were reliable， quicklv and effective tools for rapid identification of mycobacterial strains． Key words Mycobacteria PCR RFLP 中图分类号：R446．61 文献标识码：A 文章编号：1007-1245(2008)24-0005—03 以细菌学反应方法等表型特性鉴别分枝杆菌往往 1．1 材料 操作复杂，且耗时长。我们应用 16s～23srDNA间 1．1．1 菌种来源：由