舌苔细菌PCR-DGGE分析条件的建立与优化

目的 针对口腔舌苔细菌16S rDNA序列进行变性梯度凝胶电泳（denaturing gradient gel electrophoresis，DGGE）适用序列的筛选及电泳条件的优化。方法 以DGGE图谱的高分辨率为指标，选择舌苔细菌DGGE分离最适的16S rDNA高变区、电泳电压和电泳时间，并采用优化的条件分析健康青年人舌苔细菌群落的分布。结果 舌苔细菌16S rDNA V3高变区引物序列能获得更加丰富清晰的DGGE条带；基于该区，当变性剂浓度为30%~60%、电泳温度60 ℃、电压60 V和电泳时间14 h时能得到分辨率最佳的DGGE图谱。运用此优化条件对12个样本舌苔细菌群落的分析表明，舌苔微生物主要由厚壁菌门、梭杆菌门、拟杆菌门和变形菌门等组成。优化后的DGGE技术对舌苔细菌多样性的分析具有准确性、灵敏性和可重复性。结论 DGGE图谱显示，不同分析条件对图谱类型和细菌多样性指数均有所差异。利用优化的DGGE条件能有效分离舌苔细菌16S rDNA V3区序列，为口腔微生物群落结构分析提供可靠的技术支持，也为其他不同生态细菌的多样性分析提供参考。

Establishment and optimization of PCR-DGGE analysis methods for tongue coating bacteria

Objective To screen the hypervariable region of 16S rDNA sequences and optimize the experimental conditions of denaturing gradient gel electrophoresis (DGGE) for microbial analysis of tongue coating bacteria. Methods Based on the high resolution of DGGE profiles, the optimal hypervariable region, electrophoresis voltage and time were selected for the separation of tongue coating bacteria. The optimized experimental conditions was used to evaluate the bacterial communities of tongue coating of healthy youths. Results The 16S rDNA V3 hypervariable region was suitable for DGGE analysis of tongue dorsum flora due to clearer and more abundant bands. A better DGGE profile was obtained by using a combination of V3 hypervariable region at 30% - 60% of gradient range of denaturing agent, 60 ℃ of electrophoresis temperature, 60V of voltage, and 14 h of electrophoresis time. The analysis on 12 tongue-dorsum samples using the optimized conditions showed the tongue-dorsum microorganisms were mainly composed of Firmicutes, Bacteroidetes, Fusobacteria and Proteobacteria. Our optimized DGGE provided accurate, sensitive and reproducible analysis for bacterial diversity in tongue coating. Conclusion The DGGE profiles showed that different DGGE conditions present different types of maps and bacterial diversity indices. Our optimized DGGE conditions can effectively distinguish different 16S rDNA V3 sequences of tongue coating bacteria, which provide a reliable technique for the analysis of bacterial community of oral microbiome and other ecological bacteria.