维吾尔族不同龋敏感儿童变形链球菌临床分离株质子转运ATP合酶亚基cab基因遗传多态性研究

Genetic diversity of ATP synthase cab subunits amplified from Streptococcus mutans clinical isolates from Uyghur children with different caries susceptibility

目的探讨新疆维吾尔族不同龋敏感儿童变形链球菌临床分离株耐酸性及耐酸因子质子转运ATP合酶亚基cab基因多态性与耐酸力和龋敏感性的相关性，以期为新疆维吾尔族儿童龋病防治提供依据。方法选取新疆维吾尔自治区乌鲁木齐市3—5岁维吾尔族高龋（龋失补牙数≥5）和无龋（龋失补牙数=0）儿童口腔中分离的变形链球菌24和17株，采用紫外分光光度计测定540nm处吸光度值，比较不同pH条件下细菌的生长情况并筛选出16株高耐酸、17株低耐酸菌株，以特异引物从细菌组DNA扩增uneEBF，采用聚合酶链反应一限制性片段长度多态性分析技术对酶切图谱行基因分型，结合核酸测序技术比较导致基因型突变的位点。结果pH值为5．0时，高龋者变形链球菌生长能力[A值为（0．702±0．157）]较无龋者[A值为（0．372±0．123）]强，差异有统计学意义（P=0．023）；不同限制性片段长度多态性分析产生不同的基因型，测序结果证实了导致多态性出现的基因变异位点；其中内切酶AluⅠ产生的A基因型在高、低耐酸力菌株中的分布分别为7和2株，B基因型在高、低耐酸力菌株中的分布分别为9和15株，基因型分布在不同耐酸力菌株中差异有统计学意义（P=0．039）；A基因型在无龋、高龋儿童菌株中的分布分别为12和7株，B基因型在无龋、高龋儿童菌株中的分布分别为5和17株，基因型分布在不同龋敏感菌株中差异有统计学意义（P=0．009）。结论维吾尔族高龋儿童口腔中变形链球菌临床分离株具有高致龋性，且耐酸因子质子转运ATP合酶亚基联合基因uncEBF具有明显的基因多态性，不同基因型uneEBF分布与耐酸力及龋敏感性具有相关性。

Objective To investigate the aciduricity and genetic diversity of ATP synthase subunit gene uncEBF derived from Uyghur children Streptococcus mutans （Sm） clinical isolates and the relationship between the genetic diversity of ATP synthase and Sm aciduric ability and caries susceptibility. Methods Forty-one Sm strains derived from 24 caries-active individuals and 17 caries-free individuals, including 16 strains displaying high acid tolerance and 17 strains displaying low acid tolerance. Solutions of all isolated Sm with same density were made and cultured at pH 4. 0 to 7. 0 brain heart infusion（BHI） liquid. Terminal growth situation was compared. Gene uncEBF of these isolates were amplified with specific primers from Sm genomic DNA, and the polymerase chain reaction（PCR） products were analyzed by PCR-restriction fragment length polymorphism （RFLP） and sequenced. Results Aciduric ability of Sm isolated from the high caries-susceptible children were higher than that isolated from caries-free group（ P = 0. 023 ）. Alu I digested fragments of uncEBF displayed two different patterns A and B. The distributions of A and B genotype strains with different acidurance were different （ P = 0. 039 ）. A genotype included 7 strains displaying high acid tolerance and 2 strains displaying low acid tolerance; B genotype included 9 strains displaying high acid tolerance and 15 strains displaying low acid tolerance. The distributions of A and B genotype strains in different caries-sensitivity groups were different（ P = 0. 009）. A genotype included 7 high caries-susceptible strains and 12 caries-free strains; B genotype included 17 high caries-susceptible strains and 5 caries-free strain. Some of these amplified uncEBF genes from different genotype were sequenced and testified that there existed variation of Alu I recognized sites. Conelusions The high cariogenecity of Sm strains isolated from caries-active children shows a close relationship with the high aciduric ability of the isolated Sm strains. uncEBF gene of Sm F-ATPase obviously exhibits genetic diversity.