高龋者口腔中c血清型变形链球菌的基因组指纹分析

目的：确定高龋者口腔中c血清型变形链球菌的基因型分布。方法：从20例高龋者口腔中分离出c血清型变形链球菌，采用chelex法提取细菌染色体DNA，通过AP—PCR进行临床分离株的基因组指纹分析。结果：共分离获得了87株c血清型变形链球菌，不同个体所携带的变形链球菌临床分离株的基因型不同，同一个体可携带不同基因型的c血清型变形链球菌，26．7％的高龋者携带1种基因型，60．0％携带2种基因型，13．3％携带3种基因型。结论：大多数高龋者口腔中定植有2种或2种以上基因型c血清型变形链球菌。     

猛性龋儿童变链菌分离株耐酸性实验研究

目的：确定猛性龋儿童变链菌和远缘链球菌临床株的耐酸性。方法：采用紫外分光光度计比较猛性龋、非猛性龋、无龋儿童变链菌（各6株）和远缘链球菌（猛性龋儿童6株,非猛性龋和无龋儿童各3株）临床株在体外不同初始pH条件下的生长情况。结果：初始pH4．5－5．5条件下,各组变链菌生长抑制程度均明显大于远缘链球菌（P＜0．05）。初始pH4．5条件下,猛性龋儿童远缘链球菌分离株耐酸性明显强于非猛性龋和无龋儿童分离菌株（P＜0．05）。结论：远缘链球菌的耐酸性强于变链菌;猛性龋儿童远缘链球菌分离株耐酸性强。     

不同龋敏感儿童口腔不同基因型变异链球菌临床分离株在不同pH条件下产生GTF能力的比较研究

目的:探讨不同龋敏感儿童口腔变异链球菌不同基因型菌株在不同pH条件下产生GTF的能力.方法:从不同龋敏感儿童口腔变异链球菌中选取66株临床分离株,常规复苏、增菌并配制相同密度的菌悬液后,接种于不同pH值(以0.5为间隔,pH 5.0～7.0)的含2.5 g/L葡萄糖和0.05％ Tween 80的TYC培养基中厌氧培养18h;采用Somogyi法测定还原糖含量,计算酶活性.结果:在同一pH条件下,不同龋敏感儿童口腔变链菌GTF活性的差异具有统计学意义(P＜0.05),龋敏感性越高,GTF活性越大;同一龋敏感儿童口腔变链菌不同基因型菌株GTF活性的差异也具有统计学意义(P＜0.05),GTF活性随基因型的增加而增加;当pH5.5时,高龋、中龋儿童携带一种基因型菌株GTF活性显著下降(P＜0.05).结论:不同龋敏感儿童口腔变异链球菌不同基因型菌株在不同pH条件下GTF活性不同,携带基因型越多的菌株GTF活性越高.     

猛性龋儿童变链菌分离株的初始粘附能力

目的 :探讨猛性龋儿童变链菌和远缘链球菌临床分离株的初始粘附能力。方法 :采用唾液包被羟磷灰石 (SHA)及同位素标记方法 ,检测猛性龋、非猛性龋、无龋儿童变链菌 (各 6株 )和远缘链球菌 (猛性龋儿童6株 ,非猛性龋和无龋儿童各 3株 )临床株对SHA的粘附情况。结果 :各组变链菌分离株之间及各组远缘链球菌分离株之间对SHA的粘附率无显著差异 ;在无蔗糖条件下 ,各组远缘链球菌分离株对SHA的粘附百分率均低于各组变链菌 ,差异有显著性 (P <0 .0 5 )。结论 :变链菌的初始粘附能力强于远缘链球菌 ;猛性龋儿童变链菌和远缘链球菌临床株对SHA的初始粘附能力与非猛性龋及无龋儿童无差别。     

奶瓶龋儿童口腔变链菌的研究

目的：研究儿童口腔内的变链菌基因型与奶瓶龋易感性的关系。方法：取20名奶瓶龋及20名无龋儿童牙菌斑及唾液接种于TYCSB培养基计数并筛选MS,采用AP－PCR法鉴定变链菌基因型。结果：奶瓶龋组儿童唾液及菌斑变链菌计数高于无龋组;并且有两种以上基因型变链菌的比例也高于对照组（14／20vs 5/20)。结论：患有奶瓶龋的儿童不仅变链菌计数高,而且多数被两个以上基因型的MS感染。     

儿童猛性龋变链菌分离株的产酸性

目的：确定儿童猛性龋变链菌和远缘链球菌临床分离株的产酸性。方法：采用酸度计和自动气相色谱仪比较儿童猛性龋、非猛性龋、无龋变链菌（各6株）和远缘链球菌（儿童猛性龋6株,非猛性龋和无龋各3株）的临床株降低环境pH值的能力（ΔpH）和乳酸产量,并以此推测其致龋力。结果：远缘链球菌ΔpH及乳酸产量均高于变链菌,特别是在低pH水平下,二者差异更为显著（P＜0．05）。儿童猛性龋远缘链球菌分离株ΔpH和乳酸产量显著大于非猛性龋和无龋分离菌株（P＜0．05）。结论：远缘链球菌的产酸能力强于变链菌;儿童猛性龋远缘链球菌分离株较非猛性龋和无龋儿童分离菌株产酸性更强。     

猛性龋儿童变链菌分离株耐酸性实验研究

目的 :确定猛性龋儿童变链菌和远缘链球菌临床株的耐酸性。方法 :采用紫外分光光度计比较猛性龋、非猛性龋、无龋儿童变链菌 (各 6株 )和远缘链球菌 (猛性龋儿童 6株 ,非猛性龋和无龋儿童各 3株 )临床株在体外不同初始 pH条件下的生长情况。 结果 :初始 pH 4.5～ 5 .5条件下 ,各组变链菌生长抑制程度均明显大于远缘链球菌(P <0 .0 5 )。初始pH 4.5条件下 ,猛性龋儿童远缘链球菌分离株耐酸性明显强于非猛性龋和无龋儿童分离菌株(P <0 .0 5 )。结论 :远缘链球菌的耐酸性强于变链菌 ;猛性龋儿童远缘链球菌分离株耐酸性强     

高龋及无龋儿童变形链球菌分离株的蔗糖粘附能力比较

目的:探讨高龋和无龋儿童变形链球菌(变链菌)临床分离株的蔗糖依赖性粘附能力。方法:选取本实验室从10例高龋儿童和10例无龋儿童(3￣5岁)牙菌斑内分离、鉴定所得的60株变链菌临床分离株进行研究。采用紫外分光光度计,检测来自高龋儿童(dmfs≥6)的39株和无龋儿童(dmfs=0)的21株变链菌在含糖培养基中对玻壁的粘附情况。采用SPSS12.0软件包进行单因素方差分析,比较高龋儿童和无龋儿童之间牙菌斑内变链菌分离株的玻壁粘附能力。结果:在含1%蔗糖的培养基中,高龋组变链菌分离株对玻壁的粘附比平均值为(55.49+26.16)%,无龋组变形链球菌分离株对玻壁的粘附比平均值为(27.01+18.39)%,2组间差异具有显著性,P<0.01。结论:在含蔗糖环境中,分离自高龋儿童牙菌斑的变链菌株对玻壁的粘附能力高于来自无龋儿童的变链菌分离株,提示变链菌临床分离株的粘附能力与其致龋力相关。     

母子口腔中变形链球菌基因型的对照研究

目的：分析高龋、无龋儿童及其母亲口腔中变形链球菌（S．mutans）菌株的基因型，探讨S．mutans基因型与致龋活性的关系。方法：试剂盒法提取细菌染色体DNA，对20名儿童（高龋组10名，无龋组10名）及其母亲的共800株S．mutans临床分离株进行AP—PCR基因型分析。结果：高龋儿童携带的S．mutans基因型数目多于无龋儿童（P〈0．05），高龋儿童的母亲携带的S．mutans基因型数目显著多于无龋儿童的母亲（P〈0．01），高龋儿童母亲的DMFT值显著高于无龋儿童母亲（P〈0．01），高龋组和无龋组的共有基因型数目无统计学差异（P〉0．05）。结论：高龋个体比无龋个体携带更多的S．mutans基因型，个体携带的S．mutnm基因型数目与其致龋活性有关。     

不同龋敏感儿童口腔变异链球菌不同基因型临床分离株产酸力的实验研究

目的比较不同龋敏感儿童口腔变异链球菌不同基因型临床分离株对蔗糖进行酵解产生有机酸的能力。方法从不同龋敏感的3~5岁学龄前儿童口腔变异链球菌中选取66株变异链球菌临床分离株,用气相色谱法测定高龋、中龋和无龋儿童口腔变异链球菌不同基因型临床分离株产生各有机酸的量。结果不同龋敏感儿童不同基因型变异链球菌临床分离株产生有机酸的量的差异有统计学意义（P<0.05）,同一龋敏感儿童变异链球菌携带不同基因型菌株产酸量的差异也有统计学意义（P<0.05）,携带基因型数目越多的菌株其产酸量越多。变异链球菌临床分离株产生乳酸、乙酸、甲酸的量的差异有统计学意义（P<0.05）,乳酸多于乙酸和甲酸。结论不同龋敏感儿童口腔变异链球菌不同基因型菌株产酸力不同,携带基因型数目越多的菌株其产酸力越强。     

新疆维吾尔族儿童乳牙龋与变形链球菌基因型相关性的初步研究

目的 探讨新疆维吾尔族不同龋敏感儿童口腔中变形链球菌幕冈多态性与乳才龋发生的关系,以期为新疆维吾尔族儿童乳牙龋危险性预测和防治提供依据.方法 选取新疆乌鲁小齐市3～5岁维吾尔族高龋(龋失补牙数≥5)和无龋(龋失补牙数=0)儿童各17名为研究对象,收集唾液样本,轻唾一杆菌肽琼脂培养基分离培养变形链球菌.典型菌落行革兰染色...     

随意引物PCR用于口腔链球菌基因分型的初步研究

目的：探讨口腔链球菌基因分型，比较细菌相互间基因差异。方法：设计２对１０碱基随意引物，提取变链球菌、血链球菌染色体ＤＮＡ，分别用一对引物进行ＰＣＲ扩增。结果：变链球菌、血链球菌扩增的ＤＮＡ基因片段有较大区别。设计不同序列引物，扩增的ＤＮＡ片段亦不同，可以分辨不同的基因位点。结论：随意引物ＰＣＲ检测方法稳定，可作为细菌基因分型较好的方法     

Genotypic profiles by AP-PCR of streptococcus mutans in caries-active and caries-free preschoolers

Streptococcus mutans, an acidogenic and aciduric microorganism that colonizes the oral cavity is recognized as the main causal agent of dental caries. Epidemiological studies have shown a strong correlation between the number of S. mutans in the oral cavity and prevalence and incidence of caries. At present, different genotypic and phenotypic methods are known to determine the profiles of settling and epidemiological distribution of S. mutans. The aim of this study was to investigate the profiles of S. mutans isolated from children with and without dental caries by using the AP-PCR (arbitrarily primed polymerase chain reaction) and api-Zym methods. In the AP-PCR method, random DNA segments of the target bacterium are amplified with single primers of arbitrary sequence. The api-Zym system (bioMirieux, Marcy-létoile, France) is a phenotypic micro-method that allows simultaneous detection of 19 enzymatic activities from bacterial inoculum. A transversal observational study was conducted, which finally included 1203- to 5-year-old children (75 with and 45 without dental caries), who attended a preschool institution in Bogota (Colombia). S. mutans was isolated from 15 of the 45 children without dental caries (33.3%) and from 31 of the 75 children with caries (41.33%). In the 46 children, 69 S. mutans isolates were identified: 24 isolates in the 15 children without dental caries and 45 isolates in 31 children with dental caries. With api-Zym system, 36 different phenotypes were detected: 22 in the caries group and 15 in the caries-free group. The phenotype XX was present in both groups. With the AP-PCR method, 27 different fingerprinting profiles were identified: 22 for the caries group and 9 of the healthy group; the two groups of patients shared four of these genomic profiles. In conclusion, the information shows a great diversity in S. mutans genotypes and phenotypes in the population studied.     

Genotyping of Streptococcus mutans by using arbitrarily primed polymerase chain reaction in children with Down Syndrome

OBJECTIVE: The aim of this study was to compare the caries prevalence between Down Syndrome (DS) and non-DS children and to investigate the difference between the genotypes of Streptococcus mutans (S. mutans) colonized in both DS and non-DS groups. DESIGN: Sixty children with DS and 64 non-DS children aged between 7 and 12 years old were included to this study. All erupted teeth were evaluated according to the criteria recommended by the World Health Organization. Unstimulated saliva samples were carried out from the children and cultivated on S. mutans selective Tryptone-yeast-cystine (TYC) agar with 0.2 U/ml bacitracin and 15% sucrose. Molecular typing of S. mutans strains was performed by using arbitrarily primed polymerase chain reaction (AP-PCR) with OPA-05 primer. All data were analysed by using SPSS (SPSS Inc., Chicago, IL, USA) 11.0 software program for windows. RESULTS: The caries index scores were found significantly lower in DS individuals than the non-DS group (p < 0.05). The salivary S. mutans levels between DS and non-DS groups did not show significant difference (p > 0.05). The difference between dental caries and salivary S. mutans levels also was not statistically significant (p > 0.05). According to the results of the AP-PCR typing, all profiles of S. mutans which colonized in DS group were different from the control group. The relationship between these different profiles and dental caries prevalence was statistically significant (p < 0.05). CONCLUSION: The profiles of S. mutans colonized in DS group might be a reason of low caries prevalence.     

Mutans streptococci strains prevalence before and after cavity preparation during Atraumatic Restorative Treatment

Critics argue that all carious dentine is not removed from the hand-prepared cavity during the Atraumatic Restorative Treatment (ART) procedure, and that the caries process is soon resumed. The aim of this study was to determine the effectiveness of ART in removing carious tissue, by investigating the numbers of mutans streptococci and lactobacilli, with emphasis on the prevalence of Streptococcus mutans and Streptococcus sobrinus strains before, and after ART treatment of dental caries. Two microbiology samples were collected. The first sample was removed from the centre of the carious lesion at the enamel-dentine junction, and the second was collected from the centre of the hard cavity wall above the pulp, after the soft infected dentine had been manually removed. A total of 71 mutans streptococci isolates from 31 children and 40 carious teeth were subcultured, biochemically characterised and genotyped by the arbitrarily primed polymerase chain reaction (AP-PCR). Results showed a significant decrease in TVC (P<0.0001), mutans streptococci (P < 0.0001) and lactobacilli (P = 0.0002) after cavity preparation. AP-PCR identified S. mutans strains that were undetectable during biotyping, and divided clinical isolates into two main clusters. In all, 63% (45/71) of isolates from the carious lesions comprised S. mutans strains. After cavity preparation, this was reduced to 35% (25/71), of which 30% (21/71) were S. mutans and the remaining 6% (4/71) S. sobrinus strains. The number of mutans streptococci strains was below detectable levels in 19 of the prepared cavities. The significant decrease in bacteria after manual cavity preparation demonstrates the reliability of a standardized ART technique, yet the presence of S. mutans strains shows that the effectiveness of the ART procedure can vary during treatment and between dental practitioners.     

AP-PCR基因指纹用于变形链球菌传播株与非传播株的筛选

目的　应用AP-PCR基因指纹筛选变形链球菌(mutans streptococci,MS)的传播株(transmitted strains)与非传播株(nontransmitted strains),探讨影响MS传播的因素。方法　选取20对口腔中已定居有MS的儿童(3~4岁) 与他们的母亲,取牙面菌斑样本涂于轻唾-杆菌肽培养基。每人随机挑取45株分离株,提取染色体DNA,AP-PCR 基因指纹检测。结果　①从200个分离株中共分辨出45个不同的基因型,其中10位(50%)母亲和15位(75%)儿童分别带有1种类型,另5位(25%)儿童带2种类型,另10位(50%)母亲带有2种或2种以上类型(2位母亲带有5 种型),表明人群口腔中定居的MS存在基因多态性;②比较母亲与其子女MS基因型的相似性发现,20对母子中 16对(80%)有相似基因型出现,提示MS在此人群中的高传播现象;③对有传播现象的16位母亲的S.mutans进行传播株与非传播株的筛选发现,10(50%)位母亲口腔中传播株与非传播株共存,表明并非所有基因型的S.mutans 都能传播。结论　①AP-PCR基因指纹能清晰地分辨出S.mutans的传播株和非传播株;②在S.mutans的母婴传播过程中某一型菌株的优先传播是普遍存在的,进一步探讨造成这种现象的原因是很有必要的。     

幼儿猛性龋病原菌的分离鉴定

目的:确定幼儿猛性龋的优势病原菌,为其防治提供依据。方法:采用细菌分离培养、形态学、生理生化学和 DNAG+C mol%测定方法,对30名2~5岁猛性龋患儿牙菌斑菌丛进行分离鉴定,采样部位为上颌患龋乳切牙龋损部位及邻近健康釉质表面,对照组的非猛性龋和无龋儿童则采集上颌乳前牙唇面颈1/3处的菌斑。结果:猛性龋儿童龋损部位变链菌和远缘链球菌的检出率及两个采样部位菌斑标本中变链菌和远缘链球菌的检出水平均显著高于非猛性龋和无龋儿童(P<0105)。结论:变链菌和远缘链球菌为幼儿猛性龋的优势病原菌。关键词 幼儿猛性龋变链菌远缘链球菌。     

重度龋儿童远缘链球菌基因多态性研究

目的 采用随机引物聚合酶链反应(arbitrarily primed-polymerase chain reaction,AP-PCR)法初步探讨远缘链球菌在重度低龄儿童龋(severe early childhood caries,S-ECC)儿童与无龋儿童口腔中基因型分布的情况,分析其与婴幼儿龋发生之间的关系.方法 选取北京市海淀区和西城区14所幼儿园178名42～54个月龄儿童,S-ECC(患龋牙数≥5)组87例,无龋组91人.嚼蜡法采集刺激性唾液进行分离培养,典型菌落行革兰染色、生化鉴定并保种,提取基因组DNA,PCR鉴定变形链球菌和远缘链球菌,AP-PCR法对远缘链球菌临床分离株行基因型分析.结果 S-ECC组远缘链球菌检出率18%(16/87),显著高于无龋组的3%(3/91),差异有统计学意义(P＜0.01).53株远缘链球菌临床分离株共检出22种基因型,S-ECC组个体基因型为1～3种,无龋组均为1种;此外,S-ECC组3名个体间存在相同基因型的菌株.S-ECC组基因型数与龋失补牙数间存在相关性(r=0.50,P＜0.05).结论 S-ECC儿童远缘链球菌检出率明显高于无龋儿童,且菌株间存在基因多态性,个体携带基因型的种类数与其致龋性间存在相关性;远缘链球菌无关个体间存在相同基因型的菌株.     

Simple and rapid detection of Streptococcus mutans and Streptococcus sobrinus in human saliva by polymerase chain reaction

Streptococcus mutans and Streptococcus sobrinus are major pathogens causing dental caries in humans. A simple and rapid method to detect these species in human saliva simultaneously was developed using the polymerase chain reaction (PCR). Chromosomal DNA was extracted by boiling bacterial cells in lysis solution containing 1% Triton X-100. Oligonucleotide primers specific for portions of the glucosyltransferase genes (gtfB of S. mutans and gtfI of S. sobrinus) were designed. After PCR using two sets of these primers, S. mutans and S. sobrinus were specifically identified. The method was capable of amplifying DNA fragments specific for these species from chromosomal DNA extracted from 1 x 10(3) cells, or from 10 microliters of clinical saliva samples containing 1 x 10(3) colony-forming units of either streptococcal species. A second PCR, using the first PCR product as a template with newly designed internal primers, made it possible to detect 1 x 10(2) colony-forming units of either streptococcal species in 10 microliters of saliva samples. These results indicate that the PCR method developed in this study is useful for detecting S. mutans and S. sobrinus in saliva and that it can be used in epidemiological studies to evaluate the prevalence level of these organisms.