聚合酶链反应检测致龋性变形链球菌

本研究旨在应用聚合酶链反应检测致龋性变形链球菌。针对血清C型变形链球菌的spaP基因序列合成特异性引物,用聚合酶链反应(PCR)扩增spaP基因片段的方法检测变形链球菌各菌株和口腔多种常居菌。结果只有变形链球菌血清C型产生了扩增产物,呈现单一的192bpDNA条带。这种方法使过去不能检出的微量靶序列(3个cfu)得以检出,具有高度的特异性和敏感性。用本方法检测50例口腔菌斑标本,有44例呈阳性结果。提示该检测技术是一种快速、准确检测变形链球菌的新方法。     

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。     

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。     

Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction

Igarashi T, Yamamoto AA, Goto N. Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction.
Streptococcus mutans is an etiological agent in human dental caries. A method for the detection of S. mutans directly from human dental plaque by polymerase chain reaction has been developed. Oligonucleotide primers specific for a portion of the dextranase gene (dexA) of S. mutans Ingbritt (serotype c) were designed to amplify a 1272-bp DNA fragment by polymerase chain reaction. The present method specifically detected S. mutans (serotypes c, e and f), but none of the other mutans streptococci: S. cricetus (serotype a), S. rattus (serotype b), S. sobrinus (serotypes d and g), and S. downei (serotype h), other gram-positive bacteria (16 strains of 12 species of cocci and 18 strains of 12 species of bacilli) nor gram-negative bacteria (1 strain of 1 species of cocci and 20 strains of 18 species of bacilli). The method was capable of detecting 1 pg of the chromosomal DNA purified from S. mutans Ingbritt and as few as 12 colony-forming units of S. mutans cells. The S. mutans cells in human dental plaque were also directly detected. Seventy clinical isolates of S. mutans isolated from the dental plaque of 8 patients were all positive by the polymerase chain reaction. These results suggest that the dexA polymerase chain reaction is suitable for the specific detection and identification of S. mutans.     

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。     

Detection and serotype distribution of Actinobacillus actinomycetemcomitans in cardiovascular specimens from Japanese patients

Actinobacillus actinomycetemcomitans, an important pathogen in periodontitis, has also been detected in cardiovascular tissues. Sixty heart valves were collected during valve replacement surgery from 60 patients (one from each), 10 were from patients with infective endocarditis (IE group) and 50 were from patients with other valvular diseases (non-IE group). In addition, 46 samples of aneurysmal tissue were taken from 46 patients with a thoracic or abdominal aneurysm (Aneurysm group, one from each). Dental plaque samples were taken from 54 of the patients, 31 in the IE and non-IE groups and 23 in the aneurysm group. First, the distribution of A. actinomycetemcomitans in all specimens was analysed using a polymerase chain reaction method, which resulted in a positive reaction in 33 (31.1%) of the cardiovascular specimens and 25 (46.3%) of the dental plaque samples. Next, using serotype-specific sets of primers, the serotype distribution of A. actinomycetemcomitans in the cardiovascular specimens and dental plaque samples was found to be significantly different compared to dental plaque samples from Japanese subjects reported previously.     

葡萄糖浓度对变形链球菌spaP基因表达的影响

目的 观察不同浓度葡萄糖对变形链球菌初始粘附相关基因spaP表达的影响.方法 变形链球菌分别在0.2、1.0、5.0%葡萄糖条件下培养,提取总RNA,逆转录成cDNA,利用TaqMan实时荧光定量PCR技术检测不同环境条件下变形链球菌初始粘附相关基因spaP的表达情况.结果 在0.2%～5.0%葡萄糖浓度范围内,粘附较强的变形链球菌菌株spaP基因的表达随糖浓度的增加而明显增强,粘附较弱的菌株也呈现这样的趋势,但差异不具有统计学意义.粘附较强的变形链球菌菌株其spaP基因的表达总体上高于粘附较弱的菌株.结论 在一定浓度范围内(0.2%～5.0%),葡萄糖浓度升高利于变形链球菌spaP基因表达可能是其促进变形链球菌初始粘附的机制之一;变形链球菌对牙面的粘附力可能与其spaP表达量有关.     

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     

Real-time quantitative polymerase chain reaction for enumeration of Streptococcus mutans from oral samples

This study compared SYBR Green real-time quantitative PCR (qPCR) with standard plate counting for the enumeration of Streptococcus mutans in oral samples. Oral samples (n = 710) were collected from high-caries-risk children for quantification of S. mutans by qPCR using primer pairs. The S. mutans copy number was calculated with reference to a qPCR quantification cycle (Cq) standard curve and compared with the absorbance value at 600 nm of a standard suspension of S. mutans UA159. The S. mutans copy number results were evaluated in relation to standard plate count (SPC) results obtained from each sample following culture on Petri plates containing S. mutans selective media and reported as colony-forming units (CFUs). The mean S. mutans copy number calculated from qPCR was higher than the SPC CFUs (1.3 × 10(6) and 1.5 × 10(5) CFUs, respectively). The qPCR values were usually higher in individual samples and qPCR detected the presence of S. mutans 84% (231/276) of the time that the SPC did not, compared with 33% (4/12) of the time when qPCR failed to detect S. mutans and the SPC did. The qPCR technique was found to be more sensitive for detection of S. mutans from oral samples, a method that is not dependent on the viability of the sample taken and therefore is proposed as a more reliable and efficient means of quantification of S. mutans.     

不同pH值对变异链球菌ffh基因表达的影响

目的 检测变异链球菌（S. mutans）ffh基因在不同pH值条件下的表达水平,分析pH值对S. mutans ffh基因表达的影响,分析调控ffh表达的因素。方法 在不同培养时段（4 h和18 h）和不同pH值（pH值4.0~7.0）条件下培养S. mutans标准菌株UA159,提取样本,用荧光定量聚合酶链反应（qRT-PCR）法检测样本中目的基因ffh的mRNA转录水平的变化情况,分析S. mutans ffh基因在不同培养时段和pH值条件下的表达变化趋势。结果 培养4 h时,ffh基因相对表达量随着pH值的降低而减少,培养18 h时,ffh基因相对表达量随着pH值的降低而增加;相同pH值条件下,ffh基因相对表达量在培养4 h与18 h时的差异有统计学意义（P<0.05）。结论 S. mutans ffh基因的表达受细菌培养时段和pH值的影响。     

酸性环境对变形链球菌spaP基因表达的影响

目的:观察酸性环境对变形链球菌初始黏附相关基因spaP表达的影响。方法:变形链球菌分别在初始pH为5.5和7.0的条件下培养,提取总RNA,逆转录成cDNA,利用TaqMan实时荧光定量PCR技术检测不同环境条件下变形链球菌初始黏附相关基因spaP的表达。结果:pH5.5的酸性环境中变形链球菌spaP基因表达显著下调(P相似文献   

全托儿童口腔变形链球菌水平传播的初步研究

目的　通过对幼儿园全托儿童之间口腔变形链球菌水平传播的研究,为儿童龋病预防提供新的思路。方 法　选择24名3~4岁全托儿童为研究对象,其中无龋组和有龋组各12名。采用无菌牙签采集牙面菌斑,厌氧培 养48 h,根据变形链球菌的典型形态挑取单个菌落进行次代纯培养,经形态学和微量生化鉴定后将分离的菌株行 AP-PCR扩增,对来自不同个体基因型相似的菌株再进行DNA指纹分析。结果　24名受检儿童中,变形链球菌检出 率为66·7%,无龋组和有龋组检出率分别为58·3%和75·0%。经AP-PCR扩增,24名儿童口腔中检测出29个不同 基因型的变形链球菌,其中携带2个以上基因型儿童占45·8%。经DNA指纹分析,有2种基因型在11名全托儿童 口腔中有重复检出。结论　变形链球菌在全托儿童口腔中可能存在水平传播。     

儿童口腔中变形链球菌传播方式的研究

目的通过对婴儿院儿童及其母亲之间、全托儿童之间口腔中变形链球菌（简称变链菌）传播方式的研究,为儿童龋病预防提供新的思路.方法选取四川省实验婴儿院3～4岁的20名非全托儿童及其母亲和24名全托儿童作为研究对象,无菌牙签收集其菌斑样本,MSB培养基中培养48 h,每种表型菌落挑取一个代表株在TPY平板上次代纯培养,经形态学和微量生化鉴定后对分离的变链菌株行AP-PCR扩增,对不同个体出现相似基因型的变链菌株进行分析.结果44名儿童中,65.9%的儿童口腔内检出变链菌,20对母子中有10对均检出变链菌,44名儿童及20名母亲口腔中共分离出98株变链菌;均有变链菌检出的10对母子口腔中分辨出32个不同的基因型,其中7对母子有相似基因型出现;24名全托儿童口腔中共分辨出29个不同的基因型,有2种基因型在13名全托儿童口腔中有重复检出.结论变链菌在婴儿院儿童中存在水平传播及垂直传播两种传播方式.     

实时荧光定量聚合酶链反应检测远缘链球菌的实验研究

目的：建立一种实时荧光定量聚合酶链反应（real-time fluorescence quantitaive PCR,FQ PCR)方法，准确快速的定量检测唾液中的远缘链球菌。方法：在定性PCR检测的基础上设计一对特异引物，以及一条TqaMan标记的寡核苷酸探针，对己知数量的标准远缘链球菌株6715以及30名临床龋病儿童唾液样本核酸模板进行PCR扩增，并用培养鉴定的方法对30例唾液样本进行对照检测。结果：经FQ PCR检测，10^6-10^2拷贝的标准菌模板均可以用该方法检出，且循环阈值（Ct值）与起始拷贝数的对数相关性好，r=-0.999609。30例唾液样本中，13例可以检出并准确定量，培养鉴定的方法结果有11例阳性。结论：实时荧光定量PCR方法可以用于快速灵敏的定量检测远缘链球菌，比传统的培养鉴定方法显示了更大的优越性。     

Transmission of Streptococcus mutans in a group of Turkish families

BACKGROUND/AIMS: To investigate the transmission of Streptococcus mutans in a group of Turkish families using AP-polymerase chain reaction (PCR) detection. METHODS: Eight mothers who had high S. mutans levels in unstimulated saliva and 8 children aged between 2 and 3 years participated in the study. Plaque samples from each child were collected with the tips of sterile toothpicks for S. mutans counts. Although not part of the original study design, S. mutans samples were also obtained from the unstimulated saliva of the three fathers who shared the same households. Three typical isolates of S. mutans were isolated from TYCSB agar of each subject and identified by sugar fermentation tests. S. mutans ATCC 10449 was used as the reference strain. AP-PCR was conducted with OPA-05 primer. RESULTS: All of the mothers and fathers shared the similar genotypes within their children. The fathers also harbored similar genotypes to their spouses. CONCLUSION: The mothers or the fathers could be the source for the transmission of S. mutans to their children.     

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。     

中药对口腔致龋菌抑菌效果的实验研究

目的研究连翘、金银花、苦参、厚朴、虎杖、香薷、蒲公英和薄荷水煎剂对变异链球菌和白假丝酵母菌的体外抑制作用。方法将上述中药分别煎成初始质量浓度为1 g/m L的水煎剂,梯度稀释后对变异链球菌和白假丝酵母菌进行最小抑菌浓度(minimal inhibitory concentraton,MIC)测定;采用荧光分光光度计计算活菌/死菌比例,分析中药的体外抑菌效果。结果连翘、金银花、苦参和虎杖对菌液的MIC值为12.5 mg/m L,厚朴的MIC值为50 mg/m L,香薷、薄荷和蒲公英无明显抑菌效果。荧光光谱分析发现金银花、厚朴和虎杖对变异链球菌的抑制效果(死菌/总细菌)最好,分别为43.5%、49.6%和43.6%;连翘、厚朴和虎杖对白假丝酵母菌也有较好的抑菌效果,依次为33.6%、28.4%和27.7%。结论 8种中药中,金银花、厚朴和虎杖对变异链球菌的抑菌作用较好,连翘、厚朴和虎杖对白假丝酵母菌的抑菌作用较好。     

不同人群唾液中变形链球菌的定量检测及其意义

目的 建立定量检测唾液样本中变形链球菌和细菌总数的方法 ,比较变形链球菌和细胞总数的存在与不同人群中龋齿患病率的关系.方法 针对染色体DNA印迹法检测变形链球菌中出现的14 kb的HaeⅢ酶切片段,合成特异性引物(Sm~*),运用实时荧光定量聚合酶链反应(PCR)技术定量检测变形链球菌;对单纯随机抽样方法 抽取的99份唾液标本按照龋齿牙数是否为0分为龋齿组和无龋组,龋齿组72人,无龋组27人(包括从未患过龋齿者和曾患龋齿但已经过治疗和填充的无新鲜龋齿者),分别进行变形链球菌和细菌总量的检测及统计学分析.结果引物Sm~*仅对变形链球菌有特异性,定量PCR可检测的下限为0.1 μg/L;细菌总数在龋齿组和无龋组分别为51.4×10~8个/L和221.6×10~8个/L,变形链球菌占细菌总数比值分别为0.0193和0.0059,细菌总数和变形链球菌所占比值在两组间差异有统计学意义(P=0.022).结论 引物Sm~*可用于变形链球菌的定量检测;唾液中变形链球菌与总细菌数的比值与龋齿患病率相关.     

Characteristics of accumulation of oral gram-positive bacteria on mucin-conditioned glass surfaces in a model system

Strains of Streptococcus, Actinomyces and Lactobacillus were grown on glass surfaces in semi-defined medium (pH 7.0) with mucin. at a dilution rate of D = 0.1 h⁻¹, in a modified chemostat. The accumulation of cells followed four phases. In phase 1 (0-1 h). cells did not divide on the surfaces and adhesion accounted for rapid accumulation. Phase 2 (1-4 h) comprised adhesion and cell division, and accumulation slowed, cell number doubling times (Cd_t) Streptococcus , 2.7 h to 8.6 h, Actinomyces , 2.3 h to 7.5 h and Lactobacillus , 3.6 h to 3.8 h. Cell division on surfaces accounted for accumulation in phase 3 (4 h to 12 h): Cdt Streptococcus , 1.7 h to 5.2 h. Actinomyces , 2.4 h to 7.5 h and Lactobacillus , 2.2 h to 7.2 h. The biofilm stabilized in Phase 4, Cd₁ 18.5 h to 90.2 h. The numbers (10⁶ colony-forming units per cm²) of cells in stable biofilms were Streptococcus, 4.02 to 5.12, Actinomyces , 12.5 and 34.0 and Lactobacillus, 2.77. Accumulation increased (Cd₁ 0.9 h-2.7 h) when cells were exposed to glucose excess or high dilution rates and phase 2 of accumulation did not occur.     

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。