小儿常见感染菌16S-23S rRNA基因区间的分子生物学研究

Molecular biological study on 16s-23s rRNA gene interval of common infective bacteria in children

目的 探讨聚合酶链反应 (PCR)加限制性内切酶片段长度多态性 (RFLP)分析在细菌rDNA区间检测中的应用。方法 以 16S 2 3SrRNA基因区间为靶序列 ,设计引物 ,选择合适的内切酶 ,采用PCR法加RFLP法检测标准菌株及临床菌株的rDNA区间。结果 2 6株不同的标准菌株经PCR扩增后 ,分别出现一条带 ,两条带 ,三条带及多条带的不同DNA图谱 ,敏感性试验可检出 2 .5CFU的细菌 ,与人类基因组DNA、真菌及病毒无交叉反应。其中 14种菌经一步PCR扩增即可区分。另 12种菌除肺炎克雷伯氏菌与坚韧肠球菌经HinfI或AluI酶切后仍不能区分外 ,其余经其中一内切酶酶切后均能区分。 32例血培养阳性标本均扩增出与相应标准菌株相一致的图谱。结论 16S 2 3SrRNA区间基因PCR扩增加RFLP技术检测细菌rDNA区间 ,具有特异、敏感、快速、准确的特点 ,为细菌感染的病原诊断提供新的科学依据。

Objective To approach the application of PCR and RFLP in the detection of rDNA interval of bacteria. Methods A pair of primer was designed according to the gene encoding 16s-23s rRNA gene intervals, and appropriate endonucleases were chosen; PCR and RFLP analysis was used to detect the rDNA intervals of standard strains and clinical isolates. Results After PCR amplification,the different DNA maps such as one band,two bands,three bands or more bands were shown in the 26 different type strains. The sensitivity could be improved to 2.5 CFU.No signal was observed when human DNA,funguses and viruses were used as templates. Fourteen species could be distinguished immediately; whereas others must be differentiated by Hinf I or AluI digestion.All of them could be discriminated from each other apart from K.pneumoniae and E.durans. Thirty-two blood culture positive samples showed the same DNA maps as the standard DNA maps accordingly. Conclusion The technique of using PCR and RFLP in detection of 16s-23s rRNA gene interval of bacterial DNA is sensitive,rapid ,specific and accurate,and it provides a new method for clinical septicemic etiological diagnosis.