利用DGGE分析无龋和有龋儿童牙菌斑细菌组成

Profiling of microbial composition in dental plaque from caries-free and severe early childhood caries children analysed by denaturing gradient gel electrophoresis

目的:通过变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)技术分析无龋(caries free,CF)儿童和重型早期婴幼儿龋(severe early childhood caries,SECC)儿童集合牙菌斑内细菌多样性的差异。方法:无龋儿童和SECC儿童各34例,牙菌斑基因组DNA等量混合后,制备无龋和SECC儿童牙菌斑基因组库,进行全基因组放大。分别以放大前后的基因组为模板,PCR扩增16S rDNA的V2-V3区,DGGE分析,切取SECC样本的特异条带进行克隆、测序、核酸序列比对。结果:基因组在放大前后DGGE图谱一致,SECC组样本的条带数多于CF组的条带数。切取的SECC样本的4条特异条带测序后证实为3种未培养微生物和嗜沫嗜血杆菌。结论:利用DGGE技术发现了SECC儿童菌斑与CF儿童菌斑细菌组成的差异,并在SECC样本中发现了区别于CF样本的未培养微生物,这些微生物在致龋过程中发挥的作用还有待研究。

Objective：To analyze the differences of dental plaque microbial composition between caries-free（CF） and severe early childhood caries（SECC） children analysed by denaturing gradient gel electrophoresis（PCR-DGGE）.Methods：Genomic DNA was extracted from the dental plaque of CF group（n=34） and SECC group（n=34） respectively.Each DNA sample of CF and SECC group was mixed equally to construct CF genomic DNA pool and SECC genomic DNA pool,and then the two genomic DNA pools were used as template and amplified by DNA Amplification Kit.V2-V3 region of the 16S rDNA was amplified by PCR and analyzed by DGGE.Four bands in DGGE profile specifically existed in SECC lanes were excised,cloned,sequenced and searched in BLAST to find the closest relatives.Results：After whole genome amplification,DGGE showed the same DNA profile as that without amplification profile,and the bands of SECC lanes were more than those of CF lanes.Three uncultured bacteria and Aggregatibacter aphrophilus were found in SECC group.Conclusion：DGGE can be used as a tool to detect some oral bacteria,which specifically exist in SECC samples.These bacteria might play a role in the caries formation.