实时荧光定量PCR检测牙周炎患者龈下菌斑的分布

Real-time quantitative PCR for distribution of subgingival microbiota in periodontitis patients

目的应用实时荧光定量PCR技术探索侵袭性牙周炎(aggressive periodontitis,AgP)、慢性牙周炎(chronic periodontitis,CP)患者龈下菌斑中伴放线聚集杆菌(A.actinomycetemcomitans,Aa)、牙龈卟啉单胞菌(P.gingivalis,Pg)的分布规律。方法采集32例AgP、33例CP、32例牙周健康者的龈下菌斑,构建含有2种待测细菌基因片段的重组质粒,建立定量标准,采用TaqManMGB探针实时荧光定量PCR方法检测样本中细菌数量。结果本实验构建的引物及TaqManMGB探针特异性及敏感性较好。AgP组龈下菌斑Aa的检出率高于CP组(P<0.01),但2种细菌数量在组间无显著差异,两组内Pg的检出率及数量都明显高于Aa(P<0.001),另外AgP组Aa的数量、CP组Pg数量与牙周探诊深度密切相关(P<0.01及P<0.001)。结论龈下菌斑Aa的检出率可能与牙周炎类型存在一定关联,Aa可能并不是中国人群样本AgP患者龈下菌斑的优势菌,实时荧光定量PCR对牙周病学研究有广泛应用前景。

Objective Real-time quantitative PCR technique would be used to investigate the distribution and levels of A. Actinomycetemcomitans（ Aa） and P. Gingivalis（ Pg） in subgingival plaque samples from subjects with aggressive periodontitis（ AgP） and chronic periodontitis（ CP）. Methods Subgingival plaque samples were taken from 32 patients with AP,33 patients with CP and 32 periodontal healthy controls. We constructed recombinant plasmid containing species-specific gene fragments and the serial dilution of the plasmid standards,and then the bacteria were quantified by real-time PCR with TaqManMGB probes. Results This method showed accurate specificity and sensitivity,and the prevalence of Aa in AgP group was significantly higher than that in CP group,but no significant differences in the counts of both pathogens were found between periodontitis groups. The prevalence and bacterial counts of Pg were significantly higher than those of Aa within each periodontitis group（ P〈0. 001）. In addition,a significant positive correlation was found between the probing depth and the counts of Aa in AgP group（ P〈0. 01） as well as that of Pg in CP group（ P〈0. 001）. Conclusions These findings indicate the possible relation between the prevalence of Aa in the subgingival plaque and the diagnosis of periodontitis. Moreover,Aa may not be the predominant periodontopathic bacteria in individuals with AgP in Chinese. There is a great potential in the application of the real-time quantitative PCR in the periodontal research.