二重PCR检测龈下菌斑中齿垢密螺旋体感染及其与牙周炎病变程度的关系

Detection of Treponema denticola in subgingival plaques by Double PCR and the linkage between infection with the spirocheteand degree of periodontal injury

目的 建立龈下菌斑标本中齿垢密螺旋体二重PCR临床快速检测方法,了解齿垢密螺旋体感染与慢性牙周炎病变程度之间的关系。方法 采取158例不同病变程度的慢性牙周炎龈下菌斑标本,用终浓度为1％的Triton X-100 100℃水浴10 min处理标本以制备DNA模板。采用PCR检测标本中齿垢密螺旋体16S rDNA和特征性外膜蛋白基因tdpA。采用卡方检验分析齿垢密螺旋体感染与慢性牙周炎(CP)不同病变程度的关系。结果 85.4％(135/158)标本16S rDNA阳性,77.2％(122/158)tdpA阳性,14.6％(23/158)两者均阴性,未发现tdpA阳性而16S rDNA阴性的标本。重度CP龈下菌斑标本16S rDNA和tdpA检出率均高于中度cP(r2=4.03,5.71;P<0.05),中度CP龈下菌斑标本16S rDNA和tdpA检出率均明显高于轻度(X2=9.32,16.06,P<0.01)。结论 所建立的二重PCR可用于牙周炎龈下标本中齿垢密螺旋体的临床快速诊断,齿垢密螺旋体感染与牙周病变程度密切相关。

To establish a double PCR assay for detecting Treponema denticola in subgingival plaque samples and to understand the relationship between infection with the spirochete and degree of periodontal injury.Subgingival plaque samples from 158 patients with chronic periodonitis were collected.The samples were treated with 1% Triton X-100 at 100℃ water-bath for 10 min to prepare DNA templates. A PCR assay was established to simultaneous detection of T. denticola 16S rDNA and tdpA genes in the samples. Correlation between T. denticola infection and degree of periodontal injury was analysis with Chi-square test. The results showed that 85.4% (135/158) and 77.2% (122/158) of the samples were 16S rDNA and tdpA positive, respectively, and 14.6% (23/ 158) of the samples were negative for both genes. None of the positive samples could be found as positive for tdpA but negative for 16S rDNA.Both the positive detection rates of 16S rDNA and tdpA genes in the advanced samples was higher than that those in the moderate samples (X2 = 4.03,5.71;P<0.05),and so was in the moderate samples than in the mild samples (X2 = 9.32,16.06,P< 0.01). The established double PCR assay can be clinically used for fast diagnosis of Treponema denticola in subgingival plaque samples of chronic periodontitis. There is a dose relationship between Treponema denticola infection and severity of periodontitis.