Ashimoto A, Chen C, Bakker I, Slots J
Department of Periodontology, School of Dentistry, University of Southern California, Los Angeles 90089-0641, USA.
Oral Microbiol Immunol. 1996 Aug;11(4):266-73. doi: 10.1111/j.1399-302x.1996.tb00180.x.
A 16S rRNA-based polymerase chain reaction (PCR) detection method was used to determine the prevalence of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Porphyromonas gingivalis, Prevotella intermedia. Prevotella nigrescens and Treponema denticola in subgingival specimens of 50 advanced periodontitis, 50 adult gingivitis and 50 pediatric gingivitis subjects. The optimal PCR conditions were determined for each study species. Agarose gel electrophoresis of PCR products from each study species revealed a single band of the predicted size. Restriction enzyme digestion of amplicons confirmed the specificity of the amplification. PCR detection limits were in the range of 25-100 cells. No cross-reactivity with other oral micro-organisms or nonspecific amplification was observed. The prevalence by PCR in advanced periodontitis, adult gingivitis and pediatric gingivitis subjects was 30%, 14% and 14% for A. actinomycetemcomitans, 86%, 18% and 8% for B. forsythus, 74%, 52% and 78% for C. rectus, 80%, 70% and 66% for E. corrodens, 70%, 10% and 14% for P. gingivalis, 58%, 12% and 18% for P. intermedia, 52%, 20% and 22% for P. nigrescens, and 54%, 16% and 16% for T. denticola, respectively. The prevalence was higher in the advanced periodontitis group than in both adult gingivitis and pediatric gingivitis for A. actinomycetemcomitans, B. forsythus, P. gingivalis, P. intermedia, P. nigrescens and T. denticola at P < 0.01, and for E. corrodens at P < 0.05. The prevalence of C. rectus was significantly higher in the advanced periodontitis group than in the adult gingivitis group at P < 0.01. Matching results between PCR and culture occurred in 28% (B. forsythus) to 71% (A. actinomycetemcomitans) of the samples; the major discrepancy occurred in the PCR-positive/culture-negative category. Matching results between PCR and DNA probe methods were found in 84% of the subjects (B. forsythus) and 70% (P. gingivalis). Odds ratio analysis revealed statistically significant positive associations between 17 of the 28 possible combinations (P < 0.01). This study demonstrated the utility of a 16S rRNA-based PCR detection method for identifying important subgingival microorganisms. The results indicated a strong association between the study species and periodontitis. Several previously unreported symbiotic relationships were found between the 8 species tested.
采用基于16S rRNA的聚合酶链反应（PCR）检测方法，测定50例重度牙周炎患者、50例成人牙龈炎患者和50例儿童牙龈炎患者龈下标本中伴放线放线杆菌、福赛坦氏菌、直肠弯曲菌、啮蚀艾肯菌、牙龈卟啉单胞菌、中间普氏菌、变黑普氏菌和具核梭杆菌的流行情况。确定了每种研究菌种的最佳PCR条件。来自每种研究菌种的PCR产物的琼脂糖凝胶电泳显示出一条预测大小的单带。扩增子的限制性内切酶消化证实了扩增的特异性。PCR检测限在25 - 100个细胞范围内。未观察到与其他口腔微生物的交叉反应或非特异性扩增。在重度牙周炎、成人牙龈炎和儿童牙龈炎患者中，伴放线放线杆菌的PCR检出率分别为30%、14%和14%，福赛坦氏菌分别为86%、18%和8%，直肠弯曲菌分别为74%、52%和78%，啮蚀艾肯菌分别为80%、70%和66%，牙龈卟啉单胞菌分别为70%、10%和14%，中间普氏菌分别为58%、12%和18%，变黑普氏菌分别为52%、20%和22%，具核梭杆菌分别为54%、16%和16%。伴放线放线杆菌、福赛坦氏菌、牙龈卟啉单胞菌、中间普氏菌、变黑普氏菌和具核梭杆菌在重度牙周炎组中的流行率高于成人牙龈炎组和儿童牙龈炎组，P < 0.01；啮蚀艾肯菌在重度牙周炎组中的流行率高于成人牙龈炎组，P < 0.05。直肠弯曲菌在重度牙周炎组中的流行率显著高于成人牙龈炎组，P < 0.01。PCR与培养结果的匹配率在28%（福赛坦氏菌）至71%（伴放线放线杆菌）的样本中出现；主要差异出现在PCR阳性/培养阴性类别中。PCR与DNA探针方法的匹配结果在84%的受试者（福赛坦氏菌）和70%（牙龈卟啉单胞菌）中被发现。优势比分析显示，28种可能组合中的17种存在统计学上显著的正相关（P < 0.01）。本研究证明了基于16S rRNA的PCR检测方法在鉴定重要龈下微生物方面的实用性。结果表明所研究的菌种与牙周炎之间存在密切关联。在所检测的8种菌种之间发现了几种先前未报道的共生关系。
