Microbiological examination for periodontal therapy

采用GeneAmp、R序列检测系统的实时荧光定量聚合酶链式反应(Real Time PCR)技术，应用于牙周病的微生物检测。荧光来源均为带有报告和猝灭染料的TaqMan探针和SYBR Green染料。根据16S核糖体RNA基因的核苷酸序列，设计了针对伴生放线杆菌、绿色卟啉单胞菌、中间普里沃特氏菌和细菌总数的引物和探针。由于抗生素耐药基因的传播是牙周治疗中的关键问题之一，因此定量检测与四环素耐药有关的TetQ基因被纳入检测范围。TetQ基因在10~10^7个细胞(拷贝数为10~10^7)范围内呈线性关系，细菌总数在10~10^7之间呈线性关系。TaqMan和SYBR-Green两种方法在特异性、定量和灵敏度上无显著差异。然后用SYBR Green化学试剂盒检测临床菌斑样本。分别在局部给药前和给药后一周采集龈下菌斑标本。虽然大多数病例经抗生素治疗后肺炎链球菌、中间肺炎链球菌和伴放线放线菌数量有所减少，但斑块样本中含有较高水平的teq基因。实时定量聚合酶链式反应将成为牙周病微生物检测的有力工具，而抗生素耐药基因的定量监测被认为是牙周治疗的必要手段。

Real time PCR using GeneAmp^R sequence detection system; was applied to the microbiological examination in periodontal disease. Both TaqMan probe with reporter and quencher dye and SYBR Green dye were used for the source of fluorescence. The primers and probes were designed for Actinobacillus actinomycetemcomitans, Porphyromonas gigngivalis, Prevotella intermedia and total bacteria based on the nucleotide sequence of 16S ribosomal RNA gene. Since spread of antibiotic-resistance genes is one of the crucial problems in periodontal therapy, quantitative detection of tetQ gene, which confer resistance to tetracycline, was included in the examination. The detection of P. gingivalis, P. intermedia and A. actinomycetemcomitans were linear over a range of 10 to 10^7 cells (10 to 10^7 copies for tetQ gene), while the quantitative range for total bacteria was from 10^2 to 10^7. There was no significant difference between the TaqMan and SYBR-Green chemistry systems in their specificity, quantitativity and sensitivity. The SYBR Green chemistry was then used for the clinical plaque samples. Subgingival plaque samples were obtained before and one week after the local drug delivery of minocycline. Although the number of P. gngivalis, P. intermedia and A. actinomycetemcomitans decreased after the antibiotic therapy in most cases, the plaque samples contained higher level of tetQ gene. The quantitative real time PCR will be a powerful tool for microbiological examination of periodontal disease and the quantitative monitoring of antibiotic resistance gene is thought to be necessary for periodontal therapy.