Quantitative real-time PCR for Porphyromonas gingivalis and total bacteria.
Quantitative real-time PCR for Porphyromonas gingivalis and total bacteria.
复制标题
DOI：
10.1128/.38.6.2362-2365.2000
复制
发表时间：
2000
影响因子：
9.4
通讯作者：
Sharon R. Lyons;A. Griffen;E. Leys
中科院分区：
文献类型：
--
作者：
Sharon R. Lyons;A. Griffen;E. Leys
准确定量牙菌斑样品中单个细菌种类的细胞数量是了解牙周炎的细菌病因所必需的。实时PCR提供了一种灵敏、高效、可靠的定量方法。使用TaqMan系统，我们能够确定牙龈卟啉单胞菌的数量和菌斑样本中存在的细菌细胞总数。使用物种特异性引物和荧光探针，从一系列稀释的牙龈假单胞菌细胞中检测到的DNA在很大的DNA浓度范围内呈线性（相关系数= 0.96）。单独检测的牙龈假单胞菌DNA与从牙菌斑中分离的DNA混合检测的牙龈假单胞菌DNA没有差异，表明可以从临床样本中准确测定牙龈假单胞菌的水平。使用通用引物和荧光探针测定所有细菌种类的细胞总数。4种不同菌种的标准曲线结果相似（相关系数= 0.86）。从一系列人类菌斑样本中测定了牙龈假单胞菌和总细菌的水平。在来自牙周炎患者的几个样本中观察到高水平的牙龈假单胞菌，而来自健康受试者的样本中没有。实时荧光定量PCR为牙龈假单胞菌的定量提供了一种灵敏、可靠的方法。此外，它还可以测定复杂样品中存在的细菌细胞总数，从而确定牙龈假单胞菌细胞的百分比。
Accurate quantitation of the number of cells of individual bacterial species in dental plaque samples is needed for understanding the bacterial etiology of periodontitis. Real-time PCR offers a sensitive, efficient, and reliable approach to quantitation. Using the TaqMan system we were able to determine both the amount of Porphyromonas gingivalis and the total number of bacterial cells present in plaque samples. Using species-specific primers and a fluorescent probe, detection of DNA from serial dilutions of P. gingivalis cells was linear over a large range of DNA concentrations (correlation coefficient = 0.96). No difference was observed between P. gingivalis DNA alone and the same DNA mixed with DNA isolated from dental plaque, indicating that P. gingivalis levels can be determined accurately from clinical samples. The total number of cells of all bacterial species was determined using universal primers and a fluorescent probe. Standard curves using four different bacterial species gave similar results (correlation coefficient = 0.86). Levels of both P. gingivalis and total bacteria were determined from a series of human plaque samples. High levels of P. gingivalis were observed in several of the samples from subjects with periodontitis and none of those from healthy subjects. Real-time quantitative PCR provided a sensitive and reliable method for quantitating P. gingivalis. In addition, it allowed the determination of the total number of bacterial cells present in a complex sample so that the percentage of P. gingivalis cells could be determined.