Real-time PCR quantification of Treponema denticola in subgingival plaque from patients with different severities of chronic periodontitis

doi:10.3969/j.issn.2095-4344.2017.28.011

Abstract

Abstract:

BACKGROUND: Oral treponemes (especially Treponema denticola) are widely considered to play important roles in the occurrence and development of chronic peridontitis. Treponema denticola can be detected in both periodontal patients and healthy individuals, so its pathogenicity may depend on its relative numbers in subgingival plaque.
OBJECTIVE: To detect the number of Treponema denticola in subgingival plaque of patients with different severities of chronic periodontitis and to investigate the relationship between the bacterial colonization and the periodontal status.
METHODS: Subgingival plaque samples were respectively taken from 132 patients with chronic periodontitis and 40 healthy controls, admitted in the Department of Stomatology, Aviation General Hospital of Chinese Medical University from July 2015 to August 2016. Based on the clinical data, 132 patients were divided into mild (n=41), moderate (n=46), and severe (n=45) groups. The presence, relative numbers and proportion of Treponema denticola in the samples were detected by TaqMan real-time PCR assay.
RESULTS AND CONCLUSION: There was no difference in the presence of Treponema denticola in subgingival plaque between healthy and periodontal participants. The relative numbers and proportion of Treponema denticola in the periodontal patients were significantly higher than those in the healthy controls (P < 0.000 1). The quantification of Treponema denticola in different stages of periodontitis did show significant differences (P < 0.05). In addition, the bacterial level and proportion of Treponema denticola were positively correlated to the probing depths (P < 0.000 1). When the probing depth was more than 7 mm, the bacterial level and proportion of Treponema denticola were significantly increased (P < 0.05, P < 0.01). These findings suggest that Treponema denticola widely exists in subgingival plaque of human. The bacterial load and proportion of Treponema denticola are tightly related to the severity of periodontitis and probing depth. The higher bacterial level and proportion of Treponema denticola indicate a severer periodontitis. Moreover, real-time PCR possesses a broad potential in the studies on etiology, diagnosis and treatment of periodontology.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Chronic Periodontitis, Treponema, Polymerase Chain Reaction, Tissue Engineering

CLC Number:

R318

Zhang Xue-mei, Liu Pei-pei, Liu Jun, Zhang Bao-rong. Real-time PCR quantification of Treponema denticola in subgingival plaque from patients with different severities of chronic periodontitis[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(28): 4493-4498.

Figures/Tables 2

2.2 细菌实时荧光定量PCR的特异性与灵敏度应用NCBI中的Primer-BLAST程序分析引物，结果显示这对引物对齿垢密螺旋体16S rRNA序列模板是特异的；大肠杆菌DH5α稀释后用齿垢密螺旋体的引物和探针进行检测，未见扩增；实时荧光定量PCR扩增后，对PCR产物进行测序，序列经NCBI的BLAST程序分析，表明产物就是齿垢密螺旋体的基因组片段(序列同源性高达99%以上)；利用通用引物和对应探针检测大肠杆菌DH5α和所有样本，均为阳性。将齿垢密螺旋体和真细菌重组质粒进行10倍梯度稀释，可最低检出10拷贝/μL；质粒DNA浓度在102-107拷贝/μL范围内，质粒DNA浓度的对数值与Ct值具有良好的线性关系(R2=0.998)。 2.3 齿垢密螺旋体和总细菌的定量检测将齿垢密螺旋体和真细菌的重组质粒DNA连续10倍梯度稀释后，进行实时荧光定量PCR所获得的标准曲线见图1。根据标准曲线对样本中的齿垢密螺旋体和总细菌进行定量。 2.4 健康组与慢性牙周炎组齿垢密螺旋体检出率、相对检出量和所占比例的比较齿垢密螺旋体在健康组(100%)和慢性牙周炎组(100%)间的检出率无差异。健康组与慢性牙周炎组中齿垢密螺旋体相对数量和所占比例见图2，慢性牙周炎组齿垢密螺旋体的检出量、所占比例均显著高于健康组(P < 0.000 1)。健康组与轻、中和重度慢性牙周炎组齿垢密螺旋体检出量和所占比例见图3，4组间任意两组差异显著(P < 0.000 1)；轻、中和重度组的齿垢密螺旋体检出量和所占比例均高于健康组(P < 0.000 1)；中度组高于轻度组(P < 0.05)，但低于重度组(P < 0.05)。 2.5 齿垢密螺旋体细菌载量与探针深度的关系齿垢密螺旋体检出量和所占比例与性别、年龄无显著相关性，但与探诊深度呈显著正相关性(所占比例r=0.63，P < 0.000 1；检出量r=0.57，P < 0.000 1)。按照探针长度数值即5 mm、6 mm、≥7 mm进行分组，各组齿垢密螺旋体检出量和所占比例见图4。采用Kruskal-Wallis检验进行统计分析，结果表明3组间齿垢密螺旋体检出量和所占比例差异显著(P < 0.05和P < 0.01)。同时应用Mann-Whitney U检验进行两两比较，5 mm组与6 mm组的齿垢密螺旋体检出量和所占比例差异均无显著性意义(P=0.91和P=0.22)。当探诊深度≥7 mm时，齿垢密螺旋体检出量明显升高(P < 0.05)，齿垢密螺旋体所占比例也显著升高(P < 0.01)。"

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