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

使用聚合酶链反应检测和定量牙周病原体

基本信息

批准号：
06671918
负责人：
HAMACHI Takafumi
金额：
$ 0.58万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

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.

伴放线放线杆菌和牙龈卟啉单胞菌已被认为是牙周病的致病微生物。该研究项目的目的是开发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这些牙周病原体。为了特异性检测A.actinomycetemcomitans和P.gingivalis，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以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 A.actinomycetemcomitans 和 P.gingivalis 的量，一种引物用生物素标记，另一种引物在 5' 端用地高辛标记。扩增后，将生物素化的 PCR 产物施加到涂有抗生物素蛋白的微量滴定板孔中。用碱性磷酸酶缀合的抗地高辛抗体和底物对结合的PCR产物进行比色检测。比色测定的检测限仅为 50 个细菌细胞。比色测定中的吸光度值在 50 和 10^5 细菌细胞之间呈对数线性。因此，这种比色测定能够估计龈下菌斑中牙周病原体的数量。 PCR 产物的比色测定是非常有用的方法，不仅可以检测 A.actinomycetemcomitans 和 P.gingivalis 的存在，而且还可以量化龈下菌斑样品中这些牙周病原体的数量。