实时定量PCR结合DGGE检测慢性牙周炎患者不同生境中的牙龈卟啉单胞菌和齿垢密螺旋体

The quantity of Porphyromonas gingivalis and Treponema denticola in the three habitat environment of the chronic periodontitis by using real-time PCR cooperated with DGGE

目的:利用实时定量PCR结合变性梯度凝胶电泳技术(denaturing gradient gel electrophoresis,DGGE)检测慢性牙周炎患者不同生境中牙龈卟啉单胞菌、齿垢密螺旋体。方法:选取30名慢性牙周炎志愿者,分别采集龈上菌斑、龈下菌斑及唾液样本,运用DGGE技术比较分析各组样本微生物群落结构。通过偏最小二乘法(partial least squares,PLS)分析,得出不同生境相关优势条带,并对其进行克隆、测序分析,确定相应最近邻居。采用实时定量PCR技术对龈下菌斑与龈上菌斑、龈下菌斑与唾液中牙龈卟啉单胞菌及齿垢密螺旋体进行定量检测分析。结果:实时定量PCR定量检测结果显示,龈下菌斑中牙龈卟啉单胞菌及齿垢密螺旋体含量高于龈上菌斑和唾液。结论:实时定量PCR技术结合DGGE技术能更好的诠释微生物群落信息。

Objective： To detect Porphyromonas gingivalis and Treponema denticola in the different habitats of patients with chronic periodontitis by using real-time PCR combined with denaturing gradient gel electrophoresis（ DGGE）.Methods： Thirty chronic periodontitis volunteers were selected to collect supragingival plaque,subgingival plaque and saliva samples. The microbial community structure of each group was analyzed by DGGE. Then the related dominant bands in different habitats could be found through partial least squares（ PLS）. By means of cloning and sequencing analyzing them,the nearest bacteria could be determined. In addition,the quantitative detection and analysis of Porphyromonas gingivalis and Treponema denticola between supra and subgingival plaque,subgingival plaque and saliva was illustrated by using real-time polymerase chain reaction（ PCR）. Results： It is showed that the quantity of Porphyromonas gingivalis and Treponema denticola in subgingival plaque was higher than in supragingival plaque and saliva by using real-time PCR technique. Conclusion： Realtime PCR cooperated with DGGE can interpret the microbial community information better.