随意引物PCR法鉴别口腔变形链球菌的初步研究

目的 :探讨口腔变形链球菌族细菌中各个种和血清型的关系。方法 :采用随意引物PCR法对变形链球菌 (血清型c ,d ,f)及与其同源性相近的血链球菌标准菌株进行PCR扩增 ,建立口腔变形链球菌基因图谱。结果 :变形链球菌与血链球菌基因图谱差异较大 ;变形链球菌各个种基因图谱有差异 ;属于同一个种的变形链球菌不同血清型的菌株基因图谱虽相似但有差异。结论 :运用随意引物PCR法可鉴别不同种或同一种中血清型不同的变形链球菌。提示 :随意引物PCR法是一种鉴别口腔变形链球菌比较简便、准确的方法。     

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。     

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。     

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     

热敏环境培养的变形链球菌菌株与唾液SIgA的免疫反应

目的:探讨热敏环境培养变形链球菌后,与唾液分泌性免疫球蛋白A(SIgA)的免疫反应性。方法:从20例志愿者口腔中收集牙菌斑,分离临床变形链球菌,生化实验和PCR鉴定临床变形链球菌的血清型,随机引物多聚酶联反应(AP-PCR)鉴定基因型。每种基因型菌株分2组培养:实验组42℃厌氧培养;对照组37℃厌氧培养。用改良唾液收集器无菌收集非刺激性下颌下腺、舌下腺唾液,免疫印迹分析每位志愿者的唾液SIgA与自身临床菌株和参考菌株的免疫反应性。结果:20例成人共检出21种不同基因型的C型变形链球菌。实验组与对照组的变形链球菌与唾液SIgA的免疫印迹无显著差异,仅个别杂交带的密度强弱有改变。同一个体的唾液SIgA对不同基因型的菌株,出现不同的免疫印迹带;同一参考菌株与不同个体的唾液SIgA有不同的免疫印迹。结论:人群口腔变形链球菌存在多种基因型,具有遗传多样性;变形链球菌存在多种抗原成分,可以与唾液SIgA发生免疫反应,并具有个体差异;虽然变形链球菌在热敏环境中有热休克蛋白的产生,但是与唾液SIgA没有反应原性或免疫反应性较弱。     

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。     

不同基因型变异链球菌临床分离株的分离和鉴定

目的 分离鉴定变异链球菌临床分离株。方法 收集高龋患者和无龋健康人口腔中牙菌斑标本进行厌氧培养,通过细菌形态学观察、生物化学鉴定、自动微生物分析系统分析、聚合酶链反应（PCR）扩增变异链球菌基因的特异性片段表面蛋白抗原Ⅰ/Ⅱ（spaP）、葡糖基转移酶B（gtfB）和葡聚糖酶（dexA）以及基因分型等方法对获得的变异 链球菌临床分离株进行鉴定。结果 从32例临床受试者口腔中分离获得46株变异链球菌临床分离株,经生物化学, 自动微生物分析系统,变异链球菌的特异性基因spaP、gtfB和dexA的PCR扩增均鉴定为变异链球菌,基因分型发现 其中5株临床分离株的基因型与其他菌株相同。结论 获得41株不同基因型变异链球菌临床分离株。     

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.     

高龋及无龋者变形链球菌临床分离株致龋性研究Ⅰ对唾液包被羟磷灰石的粘附实验

目的：探讨来自不同龋敏感者的变形链球菌（血清型ｃ）临床分离株的粘附能力。方法：采用液体闪烁计测法测量＾３Ｈ－ＴＤＲ标记的变形链球菌临床分离株对唾液包被羟磷灰石（ＳＨＡ）的粘附。结果：同一个体所带不同基因型菌株对ＳＨＡ粘附量差异较大;高龋组个体定植的粘附能力强的菌株所占比较显著高于无龋组。结论：高龋组变形链球菌（血清型ｃ）临床菌株的高致龋力与其携带有粘附能力强的菌株密切相关。     

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     

变形链球菌表面蛋白A区遗传多态性的研究

目的:探讨变形链球菌表面蛋白A区遗传多态性与其粘附性能的关系。方法:分别选取本实验室前期工作所获得的粘附能力较强和较弱的血清c型变形链球菌临床分离株，提取全菌DNA，经PCR扩增表面蛋白A区编码基因spaP-a后，用限制性内切酶Hae班进行限制性片段长度多态性分析。结果:经Hae ]II酶切后，两组血清c型变形链球菌均出现了4种基因型，且4种基因型在两组菌株的构成情况不同。结论:血清c型变形链球菌临床分离的 spaP-a具有遗传多态性，粘附能力较强的菌株比粘附能力较弱的菌株具有更明显的异质性。     

Suppression of Streptococcus sobrinus 6715 (g) in plaques by Streptococcus mutans 32K (c)

The dental plaque of 96 healthy donors was screened for the production of such antibacterial substances as mutalipocin and bacteriocin and 192 strains of mutans streptococci isolated: 28 produced mutalipocin and 22 produced bacteriocin. Mutalipocin produced by these 28 S. mutans strains possessed similar biochemical and biological characteristics of well-characterized mutalipocin-producing strain S. mutans 32K (serotype c). When equal amounts of S. mutans 32K and S. sobrinus 6715 (g) were cultured together, cells of S. sobrinus 6715 were completely killed in 18 h. In addition, S. mutans 32K inhibited in vitro plaque formation by S. sobrinus 6715, and S. mutans 32K also eliminated in vitro plaque preformed by S. sobrinus 6715. In rat experiments, S. mutans 32K could preemptively colonize in plaque preformed by S. sobrinus 6715. On the other hand, S. sobrinus 6715 could not colonize in plaque preformed by S. mutans 32K. The results indicate that S. mutans serotype c which produces antibacterial substances is able to invade denatl plaque and replace the other mutans streptococci. This investigation offers one of the possible explanation why S. mutans serotype c is a predominant species among mutans streptococci in human plaque.     

Suppression of Streptococcus sobrinus 6715 (g) in plaques by Streptococcus mutans 32K (c)

The dental plaque of 96 healthy donors was screened for the production of such antibacterial substances as mutalipocin and bacteriocin and 192 strains of mutans streptococci isolated: 28 produced mutalipocin and 22 produced bacteriocin. Mutalipocin produced by these 28 S. mutans strains possessed similar biochemical and biological characteristics of well-characterized mutalipocin-producing strain S. mutans 32K (serotype c). When equal amounts of S. mutans 32K and S. sobrinus 6715 (g) were cultured together, cells of S. sobrinus 6715 were completely killed in 18 h. In addition, S. mutans 32K inhibited in vitro plaque formation by S. sobrinus 6715, and S. mutans 32K also eliminated in vitro plaque preformed by S. sobrinus 6715. In rat experiments, S. mutans 32K could pre-emptively colonize in plaque preformed by S. sobrinus 6715. On the other hand, S. sobrinus 6715 could not colonize in plaque preformed by S. mutans 32K. The results indicate that S. mutans serotype c which produces antibacterial substances is able to invade dental plaque and replace the other mutans streptococci. This investigation offers one of the possible explanation why S. mutans serotype c is a predominant species among mutans streptococci in human plaque.     

Arbitrarily primed polymerase chain reaction fingerprinting for the genotypic identification of mutans streptococci from humans

Determining whether two strains of bacteria are unique, identical or clonally related depends upon comparisons of phenotypic and/or genotypic traits. Individual isolates can then be grouped according to differences or similarities among those traits. One method of genotyping strains of bacteria is commonly referred to as chromosomal DNA fingerprinting. Previously, we generated chromosomal DNA fingerprints of mutans streptococci to study the transmission of this organism within families. Here, we developed and evaluated an arbitrarily primed polymerase chain reaction (AP-PCR) method for the genotypic characterization of mutans streptococci. Results were compared to those derived from the more conventional chromosomal DNA fingerprinting method. First, we showed that randomly selected clinical isolates displayed a unique banding profile by both methods; the mean similarity indices between DNA fragment patterns were 0.69 for chromosomal DNA fingerprinting and 0.74 for AP-PCR. This indicated that AP-PCR demonstrated less diversity than chromosomal DNA fingerprinting. Subsequently, we tested the agreement between chromosomal DNA fingerprinting and AP-PCR in determining genotypic similarities among 21 mutans streptococci strains obtained from 10 mother-child pairs, and 5 mutans streptococci strains from 5 fathers. The Kappa value for agreement was 0.88. We conclude that AP-PCR, which generates patterns of 8 to 12 amplicons, is capable of distinguishing strains of mutans streptococci among non-related individuals. Moreover, AP-PCR can discern both homogeneity and heterogeneity of mutans streptococci genotypes among mother and child pairs. Overall, we found that AP-PCR gave results comparable to those of chromosomal DNA fingerprinting.     

Similarity of bacteriocin activity profiles of mutans streptococci within the family when the children acquire the strains after the age of 5

It has been shown that there is a window of infectivity for mutans streptococci between the ages of 19 and 31 months, when many children acquire mutans streptococci transmitted from their mothers. Part of the children that escape this window acquire mutans streptococci at a later age. In this group, maternal transmission is expected to be less prevalent. The present study compared the bacteriocin activity profiles of mutans streptococci isolated from mothers, fathers and children when the children acquired the mutans streptococci between the ages of 5 and 11. Twelve families were randomly selected from a group of 11-year-old children who were known to have acquired mutans streptococci during this age period. From the saliva of the mothers (n = 12), fathers (n = 8) and children (n = 12) approximately 30 mutans streptococci strains were isolated. All isolates were tested twice for bacteriocin activity against 21 indicator strains with a double-layer technique. Bacteriocin activity of strains was considered to be different when the number of strains against which bacteriocin was produced differed >1 or when the width of one or more inhibition zones differed > or =4 mm. In 7/12 mother-child pairs similar profiles were found. In the 8 father-child pairs similar profiles were only found on 2 occasions. In these 2 families, all 3 ( mother, father and child) harboured strains with a similar profile. In 4/8 father-mother pairs similar profiles were found. There was no correlation between the prevalence of mutans streptococci strains, the number of indicator strains against which the strains made bacteriocin, nor the mean size of the inhibition zones and the presence of similarity of bacteriocin activity profiles of mutans streptococci within the family members. The results show that even when a child acquires mutans streptococci after the age of 5, there may be similarity between mutans streptococci in mother, father and child, indicating that transmission between the family members occurs.     

变形链球菌基因转化相关DNA片段的初步研究

目的 :研究与变链菌基因转化相关的cslA基因在临床分离株中的检出情况。方法 :实验菌株源自前期工作所获得的不同黏附力及合成IG能力的变链菌临床分离株 ,提取全菌DNA并鉴定浓度和纯度 ,PCR扩增cslA基因 ,10g/L琼脂糖电泳观察结果。结果 :6 6株变链菌临床分离株中有 5 2株扩增出cslA基因 ,检出率为 79%。cslA基因的检出率在不同黏附力及合成IG能力的菌株间无统计学差异 (P >0 .0 5 )。结论 :cslA基因在变链菌临床分离株中的分布较广泛 ;尚不能认为cslA基因在毒力不同的菌株间的分布不同。     

Latex agglutination test for detection of mutans streptococci in relation to dental caries in children.

A simple and rapid system based on a latex agglutination (LA) reaction was devised for the detection of mutans streptococci in dental plaque. Latex particles were sensitized with antibodies against whole cells of Streptococcus mutans strains MT8148 (serotype c), MT703R (e) and OMZ175 (f) and Strep. sobrinus strains B13 (d) and 6715 (g). These sensitized particles were agglutinated within a few minutes after addition of 1.0-10 ng serotype-specific antigen from the homologous organisms or the nitrous acid extract of whole cells at 10(5)-10(6) c.f.u. The LA test specifically differentiated not only mutans streptococci from the other oral streptococci but also Strep. sobrinus from Strep. mutans. The LA test was also applicable to extracts of plaque from 206 human subjects who harboured mutans streptococci. In clinical trials, the outcome of the LA test correlated significantly with the number of mutans streptococci found in plaque (p less than 0.0001), which was quantified by the selective cultivation of mutans streptococci. Furthermore, the LA test discriminated between Strep. mutans and Strep. sobrinus from human dental plaque. The sensitivity and the specificity of the LA test for detection of mutans streptococci were 78.9 and 100%. The degree of reactivity in the LA test correlated significantly with the number of decayed tooth surfaces (p less than 0.0001) and decayed and filled tooth surfaces (p less than 0.0001). These results suggest that the LA test could be useful clinically for the detection of mutans streptococci in dental plaque as well as serving as a caries-activity test.     

Horizontal transmission of Streptococcus mutans in schoolchildren

Objetive: The aim of this study was to analyze possible horizontal transmission patterns of S. mutans among 6-7-yr-old schoolchildren from the same class, identifying genotypes and their diversity and relationship with caries disease status. Study Design: Caries indexes and saliva mutans streptococci and lactobacilli counts were recorded in 42 schoolchildren. Mutans streptococci colonies were identified by means of biochemical tests and all S. mutans strains were genotyped by arbitrarily primed polymerase chain reaction. A child was considered free of S. mutans when it could not be isolated in 3 samples at 1-week intervals. Results: S. mutans was isolated in 30 schoolchildren: 20 having one genotype and 10 two genotypes. Higher mutans streptococci and caries index values were found in those with two genotypes. Five genotypes were isolated in more than 1 schoolchild and one of these was isolated in 3 schoolchildren. Our results suggest that horizontal transmission may take place. Conclusion: Schoolchildren aged 6-7 yrs may be the source of mutual transmission of S. mutans. Key words:Streptococcus mutans, Horizontal transmission, AP-PCR, genotyping     

Typing of mutans streptococci by arbitrarily primed PCR in patients undergoing orthodontic treatment

The aim of this study was to clarify the genotypic stability of mutans streptococci (MS) longitudinally during orthodontic treatment. Plaque samples were obtained from the supragingival smooth surface of the upper right teeth at four stages: prior to and after 1, 3 and 6 months of orthodontic treatment. Levels of total viable count, total streptococci and MS in dental plaque of 17 patients were recorded. Streptococci isolated from dental plaque samples were identified as MS on the basis of their morphological and biochemical properties. DNA was prepared from 713 strains of MS and the strains were then identified with polymerase chain reaction (PCR) again. Arbitrarily primed PCR (AP-PCR) fingerprinting was applied in determining the genotypes of MS. The results indicated that levels of total viable count, total streptococci and MS increased significantly after the fixed appliances were bonded. A maximum of 3 different genotypes were found in an individual. All the genotypes were found again after the application of the fixed appliances in 17 patients. A new AP-PCR typing pattern was found after the application of fixed appliances for 1 month in patient 1. That strain was not detected either prior to or after 3, or 6 months of treatment. The result indicated that the MS clones were very stable during orthodontic treatment.