Boutaga Khalil, van Winkelhoff Arie Jan, Vandenbroucke-Grauls Christina M J E, Savelkoul Paul H M
Department of Oral Microbiology, Academic Center for Dentistry Amsterdam (ACTA), Universiteit van Amsterdam, and Vrije Universiteit, The Netherlands.
J Clin Periodontol. 2006 Jun;33(6):427-33. doi: 10.1111/j.1600-051X.2006.00925.x.
For the analysis of subgingival plaque, anaerobic bacterial culture has been the gold standard for many years. Currently, molecular microbial techniques have become available to identify and quantify target organisms with high specificity and sensitivity. The technique of real-time polymerase chain reaction(RT-PCR) provides a new tool to detect oral pathogens both in oral and non-oral human infections. The aim of this study was to compare the RT-PCR and anaerobic culture for detection and quantification of six periodontal pathogens in periodontal health and disease.
Subgingival plaque samples from 259 adult patients with periodontitis and 111 healthy controls were analysed with quantitative anaerobic culture and quantitative RT-PCR for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Micromonas micros and Fusobacterium spp.
All species were more frequently isolated from patients than controls with both culture and RT-PCR. P. gingivalis, T. forsythia and M. micros appeared significant markers for disease with both techniques. P. intermedia was significantly associated with periodontitis by RT-PCR only (OR 9.7), whereas A. actinomycetemcomitans showed a significant relationship by culture only. The critical differences between culture and RT-PCR were culture-negative/PCR-positive samples which amounted to 7% for A. actinomycetemcomitans, 3% for P. gingivalis, 7% for T. forsythia, 20% for P. intermedia, 6% for M. micros, and 0.8% for Fusobacterium spp. in periodontitis patients and 12%, 3%, 2%, 35%, 14% and 0%, respectively, in the periodontally healthy group. Furthermore, periodontitis individuals had significantly higher amount of all of the test species in the subgingival plaque samples compared with healthy subjects.
RT-PCR provides a new rapid diagnostic tool and opens the opportunity to detect small numbers of oral pathogens in clinical specimens, which are under the detection limit by culture technique.
多年来，厌氧细菌培养一直是分析龈下菌斑的金标准。目前，分子微生物技术已可用于高特异性和高灵敏度地鉴定和定量目标微生物。实时聚合酶链反应（RT-PCR）技术为检测口腔和非口腔人类感染中的口腔病原体提供了一种新工具。本研究的目的是比较RT-PCR和厌氧培养法对牙周健康和疾病状态下六种牙周病原体的检测和定量。
对259例成年牙周炎患者和111例健康对照者的龈下菌斑样本进行定量厌氧培养和定量RT-PCR分析，检测伴放线放线杆菌、牙龈卟啉单胞菌、中间普氏菌、福赛坦纳菌、微小微单胞菌和梭杆菌属。
通过培养和RT-PCR检测，所有菌种在患者中分离出的频率均高于对照组。牙龈卟啉单胞菌、福赛坦纳菌和微小微单胞菌在两种技术下均表现为疾病的显著标志物。中间普氏菌仅通过RT-PCR与牙周炎显著相关（比值比9.7），而伴放线放线杆菌仅通过培养显示出显著关系。培养与RT-PCR之间的关键差异在于培养阴性/PCR阳性样本，在牙周炎患者中，伴放线放线杆菌为7%，牙龈卟啉单胞菌为3%，福赛坦纳菌为7%，中间普氏菌为20%，微小微单胞菌为6%，梭杆菌属为0.8%；在牙周健康组中分别为12%、3%、2%、35%、14%和0%。此外，与健康受试者相比，牙周炎患者龈下菌斑样本中所有检测菌种的数量均显著更高。
RT-PCR提供了一种新的快速诊断工具，为在临床标本中检测培养技术检测限以下的少量口腔病原体提供了机会。
