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目的:通过分离鉴定不同患龋情况儿童口腔中双歧杆菌的存在情况,奠定研究口腔双歧杆菌与儿童龋病发生关系的基础。方法:选取18例3~6岁高龋患儿为实验组,15例3~6岁无龋健康儿童为对照组,采集两组儿童龈上混合菌斑和口内非刺激性唾液,采用双歧杆菌特异性选择培养基对其进行专性厌氧分离培养;采用双歧杆菌特异性引物对培养后形成的单菌落进行分子生物学鉴定。结果:双歧杆菌特异性选择培养基上形成典型菌落,部分菌落通过革兰氏染色后光镜观察可见典型的Y或V状分叉,也有棍棒状和匙状;实验组双歧杆菌检出率为83.3%,对照组检出率为0,两组双歧杆菌检出率的差异有统计学意义(P﹤0.05);实验组检出双歧杆菌的样本中通过随机挑取单菌落行PCR扩增后测序,其结果为:实验组菌斑60个菌落中齿双歧杆菌占83.3%、殊形双歧杆菌占10%、栖牙双歧杆菌占6.7%;唾液样本60个菌落中齿双歧杆菌占90.0%、殊形双歧杆菌占6.7%、栖牙双歧杆菌占3.3%。结论:高龋儿童双歧杆菌的检出率均高于无龋儿童;口腔双歧杆菌中含有的菌种主要为齿双歧杆菌、殊形双歧杆菌、栖牙双歧杆菌;齿双歧杆菌在口腔双歧杆菌中所占比例最大,是高龋儿童口腔中的活跃菌种。
OBJECTIVE: To establish a basis for the study of the relationship between Bifidobacterium oralis and caries in children by isolating and identifying the existence of Bifidobacterium in oral cavity of children with different caries status. Methods: Totally 18 children aged 3 to 6 years with caries were selected as experimental group and 15 children aged 3 to 6 years as caries control group. Two groups of children were enrolled in this study. The gingival mixed plaque and oral non-irritating saliva were collected. Bacilli-specific selection medium for anaerobic isolation and culture; using Bifidobacterium specific primers for the formation of single colonies after the molecular biology identification. Results: The typical colonies were formed on the selective medium of Bifidobacterium. Some colonies showed typical Y or V bifurcation after being stained by Gram stain. Rate of 83.3%, the detection rate of the control group was 0, the detection rate of Bifidobacterium between the two groups was statistically significant (P <0.05); the experimental group Bifidobacterium samples were randomly picked single colony PCR amplification and sequencing, the results are as follows: 60 strains of bacteria in the experimental group accounted for 83.3% Bifidobacterium toothbacteria, bifidobacteria 10%, Bifidobacterium aurata 6.7%; saliva samples of 60 colonies Bifidobacteria 90.0%, bifidobacteria 6.7%, Bifidobacterium albicans 3.3%. Conclusion: The detection rate of Bifidobacterium in children with caries is higher than those in children without caries. The species of Bifidobacterium oralis mainly include Bifidobacterium bifidum, Bifidobacterium bifidum and Bifidobacterium toothpaste; Bacillus in the oral Bifidobacterium in the largest proportion, is an active caries in children with oral bacteria.