实时荧光定量PCR法检测治疗后龈下菌斑中优势菌的变化

Quantitative real-time PCR for target periodontal bacteria in subgingival plaque before and after local delivery of periocline,scaling and root planning

目的:运用实时荧光定量PCR(qRT-PCR)技术检测慢性牙周炎患者治疗前、后龈下菌斑中牙龈卟啉单胞菌(Pg)和中间普氏菌(Pi)的拷贝数,以了解qRT-PCR对2种牙周致病菌检测的敏感性和牙周治疗的疗效。方法:选取62例中、重度牙周炎患者,分别采用龈下刮治、牙周袋内盐酸米诺环素软膏(派丽奥)给药和两者综合治疗,采集治疗前、后(7d)龈下菌斑,提取细菌基因组DNA,合成针对Pg和Pi的16S rRNA基因的特异引物,运用qRT-PCR法检测Pg和Pi的拷贝数。采用SAS 9.1.3软件包对数据进行Kruskal-Wallis和Wilcoxon秩和检验。结果:Pg、Pi在不同样品组中检测到的绝对拷贝数数量分别为103～106和102～106,在慢性牙周炎患者龈下菌斑中的检出率和绝对拷贝数量显著高于健康对照组(P<0.05);3组治疗后的Pg数量比治疗前下降,其中综合治疗组和派丽奥治疗组下降率显著高于龈下刮治组。Pi的数量在派丽奥治疗组和龈下刮治组治疗后无显著减少,但在综合治疗组显著下降(P<0.05)。结论:qRT-PCR法能快速鉴定和精确定量Pg和Pi;综合治疗法比单一疗法能更有效抑制Pg和Pi。

PURPOSE： To compare the copy number of Porphyromonas gingivalis （Pg） and Prevotella intermedia （P/） in subgingival plaque before and after local delivery of periocline （2% minocycline hydrochloride ointment, MO）, scaling and root planning （SRP） by quantitative real-time PCR （qRT-PCR） and evaluate the efficacy. METHODS： Sixty-two adults with moderate to severe chronic periodontitis were selected in the study. Microbial samples were taken from pocket before and after MO and SRP （7d）. The samples were evaluated by qRT-PCR for Pg and P/. Microbiological effectiveness of treatments was assessed using Kruskal-Wallis and Wilcoxon rank-sum test. All tests were two-sided with a significance level of 0.05. All analyses were conducted with SAS 9.1.3 software package. RESULTS： The copy number of Pg and P/in subgingival plaque was 103-106 and 102-106. Bacterial loads of Pg were reduced in SPR＋ MO, SRP and MO site. The counts of P/ decreased in SRP＋ MO sites compared with those in the MO or SRP alone sites significantly （P〈0.05）. CONCLUSIONS： Quantitative real-time PCR （qRT-PCR） is used as a powerful tool with high sensitivity and specificity to quantitatively assess target periodontal bacteria. The results show that subgingival administration of MO and SRP was effective for reducing pathogenic bacteria and improving clinical outcome. Supported by 2011 National Clinical SpecialistConstruction Project; Natural Science Foundation of Zhejiang Province （LY13H140002）; Education Department Funds of Zhejiang Province（20061258） and Medical General Research Project of Zhejiang Provinee（2012KYB121）.