论文部分内容阅读
目的：通过对分枝杆菌１６ｓ～２３ｓｒＤＮＡ间隔区序列ＤＮＡＰＣＲ扩增和限制性酶切分析，评价其在分枝杆菌分类鉴定中的价值。方法：对２１种分枝杆菌和９种亲缘关系较远的非分枝杆菌和４种亲缘关系较近的非分枝杆菌的１６ｓ－２３ｓｒＤＮＡ间隔区序列ＤＮＡ进行ＰＣＲ扩增并对扩增产物进行限制性内切酶ＨａｅⅢ．ＭｓｐＩ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带，非分支杆菌一般有１～３条带。８０％的缓慢生长分枝杆菌扩增片段集中在３４０～４００ｂｐ，快速生长分枝杆菌扩增片段集中在４７０～５７５ｂｐ。单从扩增产物的琼脂糖凝胶电泳只能鉴定４２％的受试菌种。酶切结果显示：３种分枝杆菌酶切图谱没有区别，大部分分枝杆菌的酶切图谱彼此不同，而大部分非分枝杆菌不能被ＨａｅⅢ．ＭＳＰⅠ消化。结论：说明１６ｓ～２３ｓｒＤＮＡ间隔区序列ＤＮＡＰＣＲ扩增和限制性片段长度多态性分析是分枝杆菌菌种分类鉴定的一种快速、有效的方法。
OBJECTIVE: To evaluate the value of mycobacterium in the taxonomy and identification of mycobacteria by PCR amplification and restriction enzyme analysis of 16s ~ 23srDNA spacer sequences in Mycobacterium. Methods: Twenty-one mycobacteria and nine non-mycobacterial species with distant relatives and four closely related non-mycobacterial 16s-23srDNA spacer DNA sequences were amplified by PCR and amplified. Restriction enzyme HaeIII. MspI digestion reaction, analysis of different strains of amplified fragments and restriction fragment length polymorphism differences. Results: The results of PCR amplification showed that mycobacteria generally amplified 1 ~ 2 bands and non-mycobacteria generally had 1 ~ 3 bands. 80% of Mycobacterium tuberculosis amplified fragments concentrated in 340 ~ 400bp, rapid growth of Mycobacterium amplified fragments concentrated in 470 ~ 575bp. Only 42% of tested species could be identified by agarose gel electrophoresis of amplification products alone. Restriction enzyme digestion results showed that there was no difference between the three mycobacteria digestion maps, most mycobacterial digestion maps were different from each other, and most non-mycobacteria could not be Hae III. MSP Ⅰ digestion. Conclusion: It is a rapid and effective method to identify and classify mycobacteria by DNA PCR and restriction fragment length polymorphism analysis of 16s ~ 23srDNA spacer sequences.