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目的采用实时荧光定量PCR检测白天和晚上所形成牙菌斑生物膜中变异链球菌的数量,比较早晚牙菌斑中变异链球菌定植的差异。方法收集30名健康成人全口口腔洁治后白天和晚上形成的龈上菌斑,提取细菌基因组DNA。合成变异链球菌特异性引物,纯化PCR产物获得目的基因连接于pTA-TA载体,克隆于大肠埃希菌DH5α感受态细胞。选取阳性克隆测序后纯化质粒DNA,获得质粒标准品。将样本和梯度稀释的质粒标准品进行SYBR Green I实时荧光定量PCR检测,确定标准曲线,定量样本中变异链球菌DNA的拷贝数。结果早晚牙菌斑细菌基因组DNA样本的扩增曲线均在标准品的扩增曲线范围内。晚上所形成牙菌斑中定植的变异链球菌数量(对数值7.67±0.77)高于白天定植的变异链球菌数量(对数值7.25±0.62)(P=0.007)。结论牙菌斑的微生物定植存在日夜节奏变化,晚上所形成牙菌斑中变异链球菌数量多于白天。
Objective To detect the amount of Streptococcus mutans in plaque biofilm formed by day and night by real-time fluorescence quantitative PCR, and compare the difference of colonization of Streptococcus mutans in dental plaque in a day and night. Methods The gingival plaque was collected from 30 healthy adults during the day and night after oral treatment. Bacterial genomic DNA was extracted. Streptococcus mutans specific primers were synthesized, and the PCR products were purified to obtain the target gene, which was ligated into pTA-TA vector and cloned into Escherichia coli DH5α competent cells. Select the positive clones after sequencing plasmid DNA purification to obtain plasmid standard. The samples and gradient-diluted plasmid standards were subjected to SYBR Green I real-time fluorescence quantitative PCR to determine the standard curve and quantify the copy number of S. mutans DNA in the sample. Results The amplification curves of bacterial genomic DNA samples of dental plaque in the morning and evening were in the range of the amplification curve of the standard. The number of streptococcus mutans (7.67 ± 0.77 logarithmically) colonized in plaque formed at night was higher than the number of Streptococcus mutans colonized at daytime (log 7.25 ± 0.62) (P = 0.007). Conclusions The bacterial colonization of dental plaque has a rhythm of day and night, and the number of streptococcus mutans in plaque formed more in the evening than in the daytime.