双歧杆菌在儿童口腔中的分离及其分子生物学鉴定

Isdation and moleculer biology identification of Bifidobacteria from derital plague andisaliva in children

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状分又，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PER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 isolate and identify the composition and diversity of oral Bifidobacteria in the dental plaque and saliva from 33 pre-school child subjects by the traditional cultivation and 16S rDNA-PCR. Thus it can lay the founda- tion to study the association between Bifidobacteria and dental caries. Method：33 children aged from 3 to 6-year-old were selected,15 free-caries children and 18 severe-caries. Saliva and dental plaque sanlples were collected and pooled. Samples would be grown on the MTPY （Modified Trypticase Phytone Yeast extract agar）medium. And then molecular biology method was performed to identify the single colony on the MTPY medium. Result：Typical colonies were formed on the MTPY. Through gram staining visible typical Y or V bifurcate shape could be seen from optical microscope. Bifidobacteria detection rate is 83.3 % from serious carious children in the dental plaque and saliva of the experimental group. Bifidobacteria was detected 0 in the dental plaque and saliva of the free-caries children in the control group. There was significant difference between two groups （P 〈0.05 ）. In the experimental group,a total of 120 probable Bifidobacteria colonies were identified and these were Bifidobacterium dentium, Parascardovia denticolens and Scardovia inopicata. Conclusion：No matter in dental plaque or saliva of severe caries,the detection of Bifidobacteria was higher than children without caries. Thus Bifidobm＇teria in the oral cavity probably has the relationship with the severe dental caries in children. By the analysis of 16S rDNA-PCR oral Bifidobacteria mainly contain Bifidobacterium dentium, Parascardovia denticolens and Scardovia inopicata in the experi- mental analysis. Bifidobacterium dentium is active species in the children of the severe dental caries.