套式PCR检测唾液中变形链球菌及远缘链球菌

目的 探索一种快速、简便地从人类唾液中同时检测变形链球菌和远缘链球菌的方法。方法 分别以变形链球菌gtfI和远缘链球菌gtfB基因设计两组成套引物，首先用套式PCR（二次PCR）检测变形链球菌和远缘链球菌标准株和临床株，然后用套式PCR直接从唾液中检测这两种细菌。结果 变链菌（血清型c,e,f)的标准株及临床株第1次PCR扩增产物为517bp，第2次扩增产物为468bp；远缘链球菌（血清型d,g)及道勒链球菌（血清型h)的标准株及临床株第1次PCR扩增产物为712bp，第2次扩增产物为663bp;其他异种菌均不能扩增出产物，因此该PCR检测具有高度的特异性。细菌纯培养物及唾液PCR检测的敏感性分别是：第1次PCR为10^5CFU，第2次PCR为10^3CFU。结论 套式PCR能快速在人类唾液中同时检测变形链球菌和远缘链球菌。该检测方法有望运用于临床检测，对揭示两种细菌与龋病发生关系的研究具有一定价值。     

人唾液中变形链球菌的荧光定量PCR检测

目的探讨荧光定量PCR技术对人唾液中变形链球菌进行定量检测的可行性。方法从30名受试者唾液样本中提取DNA,荧光定量PCR Taqman探针法检测变形链球菌和总细菌含量。结果受试者唾液中变形链球菌的检出率为100%,含量为3.27×104~6.31×106拷贝数/mg。结论使用荧光定量PCR技术可对人唾液中变形链球菌进行快速、批量定量。     

聚合酶链反应(PCR)方法检测远缘链球菌

目的 :建立一种快速、特异、敏感的远缘链球菌PCR检测方法。方法 :根据已发表的远缘链球菌葡聚糖酶基因 (dexA)的特异片断设计一对寡核苷酸引物 ,对 12种变形链球菌群中包含a～h 8种血清型的细菌及 2 3种常见的口腔菌中进行PCR扩增 ,扩增产物电泳鉴定 ,并对特异片断进行回收 ,测序。结果 :变形链球菌群标准菌株中 ,只有远缘链球菌 (血清d、g型 )产生特异的扩增片断 ,测序结果与已发表的文献结果完全一致 ,且显示了极高的灵敏性 ,≥ 2× 10 2 个细胞均可以用该方法检出。结论 :PCR方法可以用于快速灵敏的检测远缘链球菌 ,比传统的培养鉴定方法显示了更大的优越性     

口腔血链球菌和冠心病相关性探讨

目的探讨口腔血链球菌和冠心病的相关关系.方法调查了59例患者(其中冠心病组41例,对照组18例,均通过冠状动脉造影确诊)的社会地位、吸烟、饮酒、血脂和口腔健康状况.标本用血链球菌选择性培养基(NAYS-B)培养并记录患者唾液和龈下菌斑中的血链球菌数.通过常规生化法和AP-PCR对血链球菌各菌种进行了分类鉴定.结果在逐步多元回归分析中,均衡了冠心病经典危险因素影响后,唾液和龈下菌斑中血链球菌量与冠脉狭窄程度相关,唾液中血链球菌均数对照组为(358±540)×108CFU/L,冠心病组为(435±422)×108CFU/L,F=2.72,P=0.08;前牙龈下菌斑的血链球菌均数对照组为(98±164)×107CFU/L,冠心病组为(331±484)×107CFU/L,F=5.54,P=0.02;后牙龈下菌斑的血链球菌均数对照组为(185±232)×107CFU/L,冠心病组为(352±381)×107CFU/L,F=2.86,P=0.10.冠心病组血链球菌和高氏链球菌检出率在唾液(χ2=10.937,P<0.01)和龈下菌斑中(χ2=8.582,P<0.05)明显高于对照组,而缓症链球菌和口腔链球菌检出率在两组中差异无显著性.结论冠心病患者口腔唾液和龈下菌斑中的血链球菌量明显增多可能与冠心病的发生、发展有关.     

高发龋和无龋儿童菌斑中变形链球菌及远缘链球菌的任意引物PCR检测

目的采用任意引物聚合酶链反应（arbitrarily primed-polymerase chain reaction，AP-PCR）法探讨高发龋和无龋儿童牙菌斑致龋菌的检出情况，分析变形链球菌及远缘链球菌与乳牙龋齿发生的关系。方法从20例高发龋患儿和20名无龋儿童的牙菌斑中分离、鉴定变形链球菌和远缘链球菌，以Shiroza的DNA提取方法提取细菌基因组DNA，以形态学、生化和AP-PCR的方法鉴定变形链球菌和远缘链球菌。结果AP-PCR法检测发现高发龋患儿变形链球菌和远缘链球菌的检出率分别为100％和40％，而无龋儿童两种细菌的检出率分别为75％和5％，差异有统计学意义。结论用AP-PCR法检测发现高发龋患儿变形链球菌和远缘链球菌的检出率均明显高于无龋儿童，口腔中定植的变形链球菌和远缘链球菌是乳牙龋高发的危险因素。     

PCR同时检测变形链球菌和远缘链球菌

目的:建立一种快速、简便地同时检测变形链球菌和远缘链球菌的方法。方法:以变形链球菌Dextranase基因设计引物,用PCR检测变形链球菌群细菌。结果:变形链球菌(血清型c、e、f)的PCR扩增产物为720 bp;远缘链球菌(血清型d、g)的PCR扩增产物为460 bp;道勒链球菌(血清型h)910 bp;仓鼠链球菌(血清型a),鼠链球菌(血清型b)均为1 270 bp。其它异种菌均不能扩增出产物,因此该PCR检测具有高度的特异性。从细菌纯培养物中PCR检测的敏感性为105菌落形成单位(colony-form ing un its,CFU)。结论:PCR能同时检测变形链球菌和远缘链球菌。该检测方法具有较高的敏感性和特异性,有望运用于临床检测。     

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.     

变形链球菌特异性单克隆抗体的临床初步应用

目的:分析2～4 岁儿童唾液变形链球菌水平和患龋状况的关系. 方法:选取84 名平均年龄3.8 岁的儿童进行口腔检查;采用变形链球菌特异性单克隆抗体技术检测受试儿童唾液中的变形链球菌水平,分析变形链球菌水平和患龋情况的关系;比较25 名儿童接受干预治疗前后单克隆抗体技术检测的唾液变形链球菌水平的变化. 结果:84 名受试儿童变形链球菌检出率为100%,平均密度为1.0×106/ml.25 名儿童治疗后变形链球菌水平为2×105/ml, 低于治疗前,有统计学差异(P<0.05). 结论:变形链球菌特异性单克隆抗体技术可有效观察唾液中变形链球菌水平和龋齿的变化关系,以及追踪高水平变形链球菌儿童的龋齿发展情况.     

金银花甘草人工唾液防龋的实验研究

目的 探讨金银花甘草人工唾液对龋病发生发展的抑制效果.方法 乙醇提取金银花和甘草有效成分并加入到人工唾液中,采用液体稀释法检测其抑制变形链球菌和远缘链球菌的效果;诱导摘除腮腺后的SD大鼠致龋,建立人工龋大鼠模型,分别用金银花甘草人工唾液、甲硝唑洗必泰溶液、人工唾液和蒸馏水给4组大鼠施药,采用Keyes记分法观察金银花甘草人工唾液对大鼠口腔内龋齿发生发展的抑制效果.结果 其对变形链球菌ATCC10449株和远缘链球菌OMZ65株的最小抑菌浓度分别为25、12.5 mg/ml,最小杀菌浓度分别为200、100 mg/ml.在人工龋大鼠模型中,与蒸馏水及人工唾液组相比,金银花甘草人工唾液龋齿计分明显降低,但仍高于甲硝唑洗必泰组.结论 金银花甘草人工唾液对变形链球菌和远缘链球菌生长有明显抑制作用,并可有效抑制大鼠口腔内龋齿的发生发展.     

定量聚合酶链反应检测致龋性变形链球菌

目的 创建一种临床定量检测致龋性变形链球菌的分子生物学方法。方法 采用靶基因与参照基因同步扩增法，根据变形链球菌葡聚糖酶（dexA)基因序列，设计一对特异性引物，以pET23b质粒DNA为参照基因。对196名儿童的唾液样品进行定量PCR检测并进行常规培养法的对比研究。结果 196份唾液样品定量PCR检测致龋性变形链球菌≥10^8CFU/L，唾液的检出率为91.3%。与常规培养计量法的对比符合率为94.9%。结论 变形链球菌PCR定量检测是一种早期发现龋病活性的新方法，具有快速可靠、特异性强、符合率高等特点，有广泛的临床应用前景。     

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.     

表兄链球菌的检测与儿童猛性龋的关系

目的：探讨表兄链球菌(Streptococcus sobrinus,S. sobrinus)与儿童猛性龋的关系。方法：根据前期郑州市区猛性龋调查结果,随机抽样选择3～5岁儿童66例,其中猛性龋、普通高龋及无龋组各22例。采用TYCSB培养基作变形链球菌(Streptococcus mutans, S. mutans )及S. sobrinus初步筛选,结合生理生化鉴定,并采用聚合酶链反应作最终鉴定。采用 SPSS10.0软件包对实验组与对照组S.mutans和S.sobrinus的检出率进行χ²检验,组间均数作t检验。结果： S. sobrinus在各组儿童牙菌斑中均不能脱离S. mutans而单独检出,猛性龋组S. mutans检出率高于高龋组,差异无显著性(P>0.05),而2组儿童S. sobrinus检出率的差异有显著性(P<0.05);猛性龋组与无龋组S. mutans检出率差异显著(P<0.05),S. sobrinus检出率差异显著(P< 0.01)。同时检出S. mutans和S. sobrinus的样本,其猛性龋的发生率及龋失补牙数、龋失补牙面数和平滑面龋均数与只能检出S. mutans及2种细菌均不能检出的样本的差异有显著性(P< 0.01)。结论：S. mutans与S. sobrinus是儿童猛性龋的主要致病菌,S. sobrinus与儿童猛性龋的发生有关。S. sobrinus对儿童猛性龋的发生、发展具有协同作用。     

小沟聚合物探针实时检测变形链球菌和远缘链球菌

目的　寻求一种快速检测变形链球菌(S.mutans)和远缘链球菌(S.sobrinus)的方法,研究变形链球菌群在儿童猛性龋患者口中的分布。方法　设计并合成针对S.mutans和S.sobrinus葡糖基转移酶基因(gtf)的特异性引物和小沟聚合物探针(MGB探针),对9株变形链球菌群参考菌株直接提取DNA及扩增纯培养后提取DNA分别进行检测,比较二者检测结果的异同。采集92例猛性龋儿童菌斑样本,用MGB探针进行实时检测。结果　采用MGB 探针可以特异性地鉴别S.mutans和S.sobrinus,直接检测和扩增纯培养后的定性检测结果完全一致,前者荧光出现的时间略迟。92例猛性龋患儿中S.mutans检出率为96.7%,S.sobrinus检出率为32.6%,所有检出S.sobrinus的菌斑样本均可检出S.mutans。结论　采用特异性MGB探针方法可以对菌斑中S.mutans和S.sobrinus进行实时检测, 提高检测效率。     

Susceptibility of Streptococcus mutans and Streptococcus sobrinus to antimicrobial agents after short-term oral chlorhexidine treatments

Effects of three different types of short-term applications (1–3 limes during 1 week) of chlorhexidine (1 or 40%) on the susceptibility of 863 clinical isolates of Streptococcus mutans and 53 isolates of Streptococcus sobrinus from 58 subjects were studied. Chlorhexidine-resistant isolates were not found either before or after the treatment. The minimum inhibitory concentrations (MICs) to chlorhexidine of all isolates of S. mutans were ≤1 μg/ml, and of S. sobrinus ≤2 μg/ ml. S. mutans and S. sobrinus were also susceptible to ampicillin, penicillin, cefuroxime, and tetracycline. In conclusion, different short-term chlorhexidine regimens do not induce resistance in S. mutans or S. sobrinus and, furthermore, these species have so far retained their susceptibility to common antibiotics.     

In vitro inhibitory effects of Polygonum cuspidatum on bacterial viability and virulence factors of Streptococcus mutans and Streptococcus sobrinus

OBJECTIVES: Polygonum cuspidatum has been used in Korean folk medicine to improve oral hygiene. This study was performed to evaluate the effects of methanol extract from root of P. cuspidatum (MEP) on bacterial viability and the virulence factors of Streptococcus mutans and Streptococcus sobrinus. METHODS: To test the effects of MEP on bacterial viability, we determined the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against 20 bacterial strains, including S. mutans and S. sobrinus, using a micro-dilution assay. In case of S. mutans and S. sobrinus, the assays for time-kill and bacterial growth rate at sub-MIC concentrations were also performed. To determine effects of the extract on the virulence factors of S. mutans and S. sobrinus, the assays for sucrose-dependent adherence, water-insoluble glucan formation, glycolytic acid production, and acid tolerance were performed at sub-MIC levels. Phytochemical analysis for constituents of MEP was carried out. RESULTS: MEP showed a broad antibacterial range (MIC 0.5-4 mg/ml). The MBC was two to four times higher than the MIC. The time-kill curves showed S. mutans and S. sobrinus were significantly killed after 1h of incubation. At sub-MIC levels, doubling times of S. mutans and S. sobrinus dose-dependently increased up to 211% and 123%, respectively. At sub-MIC levels, MEP also showed inhibitory effects on the virulence factors of S. mutans and S. sobrinus in a dose-dependent fashion. Phytochemical analysis revealed the presence of alkaloids, sterol/terpenes, tannins, flavonoids, and carbohydrates. CONCLUSION: These data indicate that MEP has inhibitory effects on bacterial viability at higher concentrations (> or =MIC) and the virulence factors of S. mutans and S. sobrinus at sub-MIC concentrations, suggesting that it might be useful for the control of dental plaque formation and subsequent dental caries formation.     

The earlier the colonization by mutans streptococci, the higher the caries prevalence at 4 years of age

Seventy-eight 4-year-old children, examined earlier for the presence of mutans streptococci at 4-month intervals from 15 months of age, were recalled for recording of dental caries and salivary sampling. The saliva samples were analysed for the presence of mutans streptococci, including the species Streptococcus mutans and Streptococcus sobrinus and lactobacilli. The results showed that the earlier the mutans streptococci had been detected in the children, the higher the caries experience. Eighty-nine percent of the children colonized at 2 years of age had experienced caries and had a mean dfs of 5.0 compared with 25% of the non-colonized 4-year-olds with a mean dfs of 0.3. S. mutans was the predominant species. S. sobrinus was usually found in combination with S. mutans , except in 2 children where S. sobrinus was the only species detected. More children with multiple species had higher numbers of total mutans streptococci and a tendency to higher caries prevalence than children with only S. mutans.