Detection and quantification of periodontal pathogens by the use of polymerase chain reaction

伴生放线杆菌和牙龈卟啉单胞菌被认为是牙周病的致病微生物。本研究的目的是建立一种基于聚合酶链式反应(PCR)的方法来检测和定量牙周菌斑样本中的这些病原菌。为了对伴生放线杆菌和牙龈假单胞菌进行特异性检测，分别选择了白毒素基因和胶原酶基因作为靶序列。合成了两对寡核苷酸引物，分别扩增出白毒素基因片段(396bp)和胶原酶基因片段(414bp)。扩增后，用琼脂糖凝胶电泳法对扩增产物进行分析。聚合酶链式反应方法能检测到多达50个细菌细胞。为了定量测定伴生放线菌和牙龈假单胞菌的数量，分别用生物素和地高辛标记了两条引物的5‘端。扩增后，将生物素化的聚合酶链式反应产物应用于包被亲和素的微量平板。用碱性磷酸酶偶联的抗地高辛抗体和底物比色检测结合的PCR产物。该比色法的检出限低至50个细菌细胞。比色法测定的吸光度在50~10^5个细菌细胞之间呈对数线性关系。因此，该比色法能够估计龈下菌斑中牙周病原菌的数量。聚合酶链式反应产物的比色分析不仅是检测伴生放线菌和牙龈假单胞菌的常用方法，而且还可以定量检测这些牙周病原体在龈下菌斑中的数量。

Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis has been implicated as causative organisms of periodontal disease. The aim of this research project was to develop a polymerase chain reaction (PCR) based method for detection and quantification of these periodontal pathogens in subgingival plaque samples. For specific detection of A.actinomycetemcomitans and P.gingivalis, leukotoxin gene and collagenase gene, respectively, were selected as the target sequence. Two pairs of oligonucleotide primers were synthesized to amplify the leukotoxin gene fragment (396bp) and the collagenase gene fragment (414bp) . Following PCR amplification, PCR products were analyzed by agarose get electrophoresis. PCR method was able to detect as few as 50 bacterial cells. To quantify the amount of A.actinomycetemcomitans and P.gingivalis, one primer was labeled with biotin and the other one was labeled with digoxigenin at 5'ends. Following amplification, the biotinylated PCR products were applied to a microtiter plate well percoated with avidin. The bound PCR products were detected colorimetrically with alkaline phosphatase conjugated anti-digoxigenin antibody and substrate. The detection limit of the colorimetric assay was found to be as few as 50 bacterial cells. The absorbance value in the colorimetric assay were log-linear between 50 and 10^5 bacterial cells. Therefore, this colorimetric assay was able to estimate the amount of periodontal pathogens in subgingival plaque. The colorimetric assay of the PCR product is very usuful method not only to detect the presence of A.actinomycetemcomitans and P.gingivalis but also to quantify the amount of these periodontal pathogens in subgingival plaque samples.