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

伴放线放线杆菌和牙龈卟啉单胞菌被认为是牙周病的致病菌。本研究的目的是建立一种基于聚合酶链反应（PCR）的方法，用于检测和定量龈下菌斑样本中的牙周病原体。为了特异性检测伴放线菌和牙龈卟啉单胞菌，分别选择白细胞毒素基因和胶原酶基因作为靶序列。合成两对寡核苷酸引物，分别扩增白细胞毒素基因片段（396bp）和胶原酶基因片段（414bp）。PCR扩增后，通过琼脂糖凝胶电泳分析PCR产物。PCR方法能够检测到少至50个细菌细胞。为了定量伴放线菌和牙龈卟啉单胞菌的量，一个引物用生物素标记，另一个引物在5 '端用地高辛标记。扩增后，将生物素化的PCR产物施加到用抗生物素蛋白预包被的微量滴定板孔中。结合的PCR产物用碱性磷酸酶偶联的抗地高辛抗体和底物进行比色检测。发现比色测定的检测限少至50个细菌细胞。比色测定中的吸光度值在50和10^5个细菌细胞之间呈对数线性。因此，本比色法能够估计龈下菌斑中牙周致病菌的数量。PCR产物的比色分析不仅可以检测伴放线放线菌和牙龈卟啉单胞菌的存在，而且可以定量龈下菌斑样品中这些牙周病原体的量。

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.