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目的建立PCR方法对正畸治疗患者口腔中的伴放线菌聚集杆菌(Aa)及毒力因子细胞致死性膨胀毒素进行检测,并与牙龈指数进行相关性分析。方法 选择55例戴入矫治器后产生牙龈炎性反应患者为矫治组,34例未带矫治器的牙周健康者为对照组,49例口腔内科就诊的成人牙周炎患者为成人牙周炎组。记录牙龈炎症指数,用无菌纸尖法分别采集牙周袋最深处及龈沟内液标本进行细菌DNA提取及PCR反应。结果以16S rDNA引物PCR扩增138例患者临床标本,共扩增出Aa 44株;其中正畸治疗组29株;对照组5株;成人牙周炎组10株。用Spearm an等级相关分析矫治组Aa检出率与牙龈指数G I之间存在明显的正相关关系(P<0.01)。矫治组Aa的检出率明显高于牙周炎组和对照组(P<0.01);而牙周炎组与对照组之间的Aa检出率无明显差异(P>0.05)。44例PCR扩增阳性的患者平均年龄为16.23岁,而Aa阴性的患者年龄平均为38.73岁,二者具有明显差异(t=3.598,P<0.01)。以CDT引物直接扩增44例Aa16S rDNA阳性的临床标本中的CDT基因片段,13例为阳性,其阳性率为29.54%,其中扩增出667bp的CDT1型3例,扩增出1893bp的CDT2型10例。结论 Aa在青少年患者尤其在带矫治器的青少年患者发病中起到重要的作用,但对成人牙周炎的致病不起主要作用。CDT扩增产物有两种,但检出数量较少,还有待于进一步研究。
OBJECTIVE: To establish a PCR method for the detection of lethal dilatant toxins in the mouth of patients with orthodontic treatment of Actinomyces pullulans (Aa) and virulence factors, and to analyze the correlation with gingival index. Methods 55 patients with gingivitis were selected as orthodontic group after wearing orthodontic appliance. 34 healthy people with periodontal disease without orthodontic appliances were selected as control group. 49 adult patients with periodontitis treated by oral medicine were adult periodontitis group. Gingival inflammation index was recorded. Bacterial DNA extraction and PCR reaction were performed on the specimens of the most probable periodontal pocket and gingival sulcus with aseptic tips. Results A total of 138 Aa strains were amplified by polymerase chain reaction (PCR) with 16S rDNA primers. A total of 44 strains of Aa were amplified, of which 29 were orthodontic treatment group, 5 were control group and 10 were adult periodontitis group. There was a significant positive correlation between detection rate of Aa and gingival index G I in Spearman rank correlation analysis (P <0.01). The detection rate of Aa in the correction group was significantly higher than that in the periodontitis group and the control group (P <0.01). There was no significant difference in the detection rate of Aa between the periodontitis group and the control group (P> 0.05). The average age of 44 patients with positive PCR amplification was 16.23 years, while the average age of patients with Aa negative was 38.73 years old, with significant difference (t = 3.598, P <0.01). The CDT gene fragment of 44 Aa16S rDNA positive clinical samples was directly amplified by CDT primers, 13 were positive and the positive rate was 29.54%. Among them, 667bp of CDT1 was amplified in 3 cases and 1893bp of CDT2 10 cases. Conclusions Aa plays an important role in the onset of juvenile patients, especially in juvenile patients with appliances, but it does not play a major role in the pathogenesis of adult periodontitis. There are two kinds of CDT amplification products, but the number of detected small, yet to be further studied.