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目的建立多重PCR方法检测正畸治疗患者口腔中伴放线放线杆菌(Aa)、福塞氏类杆菌(Bf)、具核梭杆菌(Fn)、牙龈卟啉单胞菌(Pg)4种细菌的存在,并与牙龈指数进行相关性分析。方法选择55例正畸治疗至少2个月的青少年患者为矫治组,34例未带矫治器的牙周健康者为对照组,记录牙龈指数,分别采龈沟液标本进行细菌DNA提取及聚合酶链式反应;同时,以细菌的厌氧培养和生化反应鉴定为标准进行对比验证,并做了PCR的敏感性和特异性实验。结果建立的多重PCR最低可检测出细菌DNA1pg,约相当于20个Aa、Fn和Pg,80个Bf;能扩增出Aa、Bf、Fn、Pg4种参考菌株的目的条带,而对大肠杆菌的扩增没有目的条带;多重PCR法与常规细菌培养法对细菌的检测阳性率较为一致(P>0.05);Aa、Fn、Pg在两组间有明显差异(P<0.05),对Bf的检测未见明显差异(P>0.05);Aa、Fn、Pg的阳性率与牙龈指数之间呈正相关(P<0.01);而Bf与牙龈指数之间无相关性(P>0.05)。结论建立的多重PCR有较高的敏感性和特异性,可同时检测龈沟液中4种常见牙周致病菌,观察固定矫治器使用过程中牙周细菌的变化。
Objective To establish a multiplex PCR method for the detection of four kinds of Aa, Bf, Fn and Pg in the oral cavity of patients undergoing orthodontic treatment. The presence of bacteria, and gingival index correlation analysis. Methods Fifty-five adolescents with orthodontics for at least 2 months were selected as treatment group and 34 healthy periodontal healthy persons without orthodontics as control group. Gingival index was recorded. Bacterial DNA was extracted from gingival crevicular fluid and polymerase Chain reaction; the same time, bacterial anaerobic culture and biochemical reactions identified as a standard comparison and verification, and do PCR sensitivity and specificity experiments. Results The results showed that 1μg of bacterial DNA was detected by PCR, which was equivalent to about 20 Aa, Fn and Pg, 80 Bf. Aa, Bf, Fn and Pg could amplify the target bands of 4 reference strains of Escherichia coli (P> 0.05). There were significant differences in Aa, Fn and Pg between the two groups (P <0.05), and the positive rate of Bf (P> 0.05). The positive rates of Aa, Fn and Pg were positively correlated with gingival index (P <0.01). There was no correlation between Bf and gingival index (P> 0.05). Conclusion The established multiplex PCR has high sensitivity and specificity. It can detect four kinds of common periodontal pathogens in gingival crevicular fluid at the same time, and observe the changes of periodontal bacteria in fixed appliance.