16S rDNA克隆文库法与PCR-DGGE法在口腔菌群分析中的对比研究

摘要

Objective To compare the efficiency of 16S rDNA clone library versus PCR-DGGE method in detecting oral microbial diversity.Methods Bacterial samples were collected from root canals of 6 patients with retrograde pulpitis.Total DNA was extracted.The universal primers 27F/1492R and HAD-1/2 were used to amplify the full length or V2-V3 region of 16S rDNA,respectively.The PCR products were separated through agarose gel electrophoresis or denaturing gradient gel electrophoresis (DGGE).The bands were cloned and sequenced.The bacteria were identified by aligning the sequences in ribosomal database.Results One band of 1.5 kb corresponding to the full length of 16S rDNA was separated through agarose gel electrophoresis.DGGE fingerprints showed 6-12 bands of 239 bp corresponding to V2-V3 region of 16S rDNA.A total of 8-15 and 6-12 kinds of bacteria were detected in one sample by 16S rDNA and PCR-DGGE method,respectively.Conclusion Compared with PCR-DGGE method,16S rDNA clone library method is a more sensible and specific technology in detecting bacterial composition in the flora.%目的 比较16S rDNA克隆文库法与变性梯度凝胶电泳联合PCR法(PCR-DGGE)在口腔微生物多样性研究中的检测效率. 方法 采集6例逆行性牙髓炎患者根管细菌样本,提取细菌总DNA,采用细菌通用引物27F/1492R、HAD-1/2分别扩增16S rDNA的全长或V2-V3区域,PCR产物分别经琼脂糖凝胶电泳和DGGE分离,纯化目的条带后克隆测序.测序结果与数据库序列比对,鉴定细菌组成. 结果 琼脂糖电泳分离到16S rDNA约1.5 kb条带,每个样本检测到8-15种细菌;DGGE分离到V2-V3区约含6-12条位置不同的239 bp谱带,每个样本检测到6-12种细菌. 结论 与DGGE法相比,16S rDNA克隆文库法测得的菌群更全面,敏感性和特异性更高.