早晚牙菌斑变异链球菌定植差异的定量PCR检测  

Difference in Streptococcus mutans colonization in dental plaque between day and night by real-time fluorescence quantitative PCR

作　　者：刘红春[1] 陈敏珊 欧阳勇[1] 涂家珍[1] 江山 

机构地区：[1]中山大学光华口腔医学院.附属口腔医院广东省口腔医学重点实验室,广东广州510055 [2]广州薇美姿个人护理用品有限公司,广东广州510665

出　　处：《中国微生态学杂志》2013年第6期659-662,共4页Chinese Journal of Microecology

摘　　要：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 compare the difference in Streptococcus mutans（S.mutans） colonization in dental plaque between day and night,by detecting the amounts of S.mutans DNA copies using real-time fluorescence quantitative polymerase chain reaction（qPCR）.Methods Samples of supragingival plaques from 30 healthy adults after a complete prophylaxis formed during day and night,respectively,were obtained.The total genomic DNA of bacteria was extracted and specific primers for S.mutans were designed.Target gene purified from PCR products was ligated into pTA-TA vector,and the vectors were transformed into competent cells of Escherichia coli DH5α.Positive recombinant plasmids were sequenced and plasmid DNA was purified.Both sample DNA and five serial dilutions of plasmid DNA from 103 to 108 were tested in triplicate using a qPCR method.Standard curves were obtained and the copy numbers of S.mutans in the samples were quantified.Results The amplification plots obtained using SYBR Green I assay for S.mutans were in the dimensions of those for plasmid DNA.S.mutans copy numbers in plaque formed during night（in lg,7.67±0.77） were higher than those formed during day（in lg,7.25±0.62）（P=0.007）.Conclusion The colonization patterns of S.mutans differ between day and night.The amounts of S.mutans in dental plaque formed during night are higher than those during day.

关 键 词：实时荧光定量PCR 牙菌斑 变异链球菌 

分 类 号：R378.12[医药卫生—病原生物学]