应用实时荧光定量PCR定量检测慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌

Real-time polymerase chain reaction for quantification of Porphyromonas gingivalis in periodontal patients

目的检测慢性牙周炎患者和牙周健康人龈下菌斑中牙龈卟啉单胞菌、总菌量和牙龈卟啉单胞菌所占比例,探讨牙龈卟啉单胞菌与牙周炎发生发展的关系。方法采集经常规PCR方法检测牙龈卟啉单胞菌为阳性的76例慢性牙周炎患者和25例牙周健康者的龈下菌斑,应用TaqMan实时荧光定量PCR方法定量检测样本中牙龈卟啉单胞菌、总菌量;构建含有牙龈卟啉单胞菌和真细菌扩增片断的重组质粒,建立定量标准。结果本研究设计的引物和探针具有良好的特异性及敏感性。病变位点龈下菌斑中牙龈卟啉单胞菌数量和总菌量均比健康位点高(P<0.001),两组位点牙龈卟啉单胞菌在菌斑中的比例没有差异(P>0.05);细菌数量与探诊深度间存在显著正相关关系(P<0.001),不同的探诊深度牙龈卟啉单胞菌所占比例无统计学差异(P>0.05)。结论龈下菌斑中牙龈卟啉单胞菌的数量水平及细菌总量与牙周状况、牙周炎发展有密切关系,实时荧光定量PCR对牙周病学研究具有广泛的应用前景。

Objective To detect the number of P. gingivalis and total bacteria in subgingival specimens from the patients with chronic periodontitis and from the periodontally - healthy subjects using TaqMan real - time polymerase chain reaction （PCR）, and to investigate the relationship between the bacterial load and the periodontal status. Methods Subgingival plaque samples were taken from 76 patients with chronic periodontitis and 25 periodontally - healthy controls （positive of P. gingivalis by conventional PCR）, respectively. The numbers of P. gingivalls , total bacteria and the proportion of P. gingivalis in the samples were detected with the TaqMan real - time PCR. Quantification for the bacteria was performed with the species - specific primers/probes, universal primer/probe and the serial dilution of the plasmid standards. Results P. gingivalis and total bacteria were significantly higher in diseased sites of the patients than in healthy sites of the controls （ P 〈0.001）, There was no difference between the proportion ofP. gingivalis in subgingival plaque of the diseased sites and healthy sites（ P 〉0.05）. In addition, a significant correlation was found between the total bacterial level and the probing depths. However, there was no difference between the proportion of P. gingivalis in subgingival plaque with probing depth（ P 〉 0.05 ）. Conclusion The numbers of P. gingivalis in the subgingival plaque are closely related to the periodontal status. There is a great potential in the application of the real - time pelymerase chain reaction in the periodontological research.