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。     

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。     

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.     

Absence of Helicobacter pylori in the oral cavity of 10 non‐dyspeptic subjects demonstrated by real‐time polymerase chain reaction

Helicobacter pylori plays a significant role in gastric disease. However, the presence of this bacterium in the oral cavity remains controversial. The aim of the present study was to detect and quantify H. pylori in 29 different sites of the oral cavity in non‐dyspeptic subjects by means of real‐time polymerase chain reactions (PCR). Ten subjects without gastric symptoms were studied. Samples from unstimulated saliva, three sites of the tongue, oral mucosa, and 12 sites of both supragingival and subgingival plaque were collected from each subject. DNA was extracted from the oral samples and analysed for the presence of H. pylori by real‐time PCR (LightCycler^®) using JW23/22 primers which targeted the 16S rRNA gene. DNA from H. pylori DSM 4867 was used as a positive control. Amplification efficiency for the LightCycler^® 2.0 runs ranged from 1.8 to 2.4. Melting curve analysis identified all the positive control capillaries, which contained H. pylori reference DNA, as a single and narrow peak at a melting temperature between 84.5 and 84.9°C. All the negative control capillaries with no template control and the 29 oral samples from each subject showed either no melting peaks or broad melting peaks below 80°C, which were considered as primer dimers. Therefore, H. pylori was not detected from any of the 290 oral samples. As a conclusion, H. pylori seems not to be permanently present in the oral cavity of a non‐dyspeptic population.     

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。     

Absence of Helicobacter pylori in the oral cavity of 10 non-dyspeptic subjects demonstrated by real-time polymerase chain reaction

Helicobacter pylori plays a significant role in gastric disease. However, the presence of this bacterium in the oral cavity remains controversial. The aim of the present study was to detect and quantify H. pylori in 29 different sites of the oral cavity in non-dyspeptic subjects by means of real-time polymerase chain reactions (PCR). Ten subjects without gastric symptoms were studied. Samples from unstimulated saliva, three sites of the tongue, oral mucosa, and 12 sites of both supragingival and subgingival plaque were collected from each subject. DNA was extracted from the oral samples and analysed for the presence of H. pylori by real-time PCR (LightCycler) using JW23/22 primers which targeted the 16S rRNA gene. DNA from H. pylori DSM 4867 was used as a positive control. Amplification efficiency for the LightCycler 2.0 runs ranged from 1.8 to 2.4. Melting curve analysis identified all the positive control capillaries, which contained H. pylori reference DNA, as a single and narrow peak at a melting temperature between 84.5 and 84.9 degrees C. All the negative control capillaries with no template control and the 29 oral samples from each subject showed either no melting peaks or broad melting peaks below 80 degrees C, which were considered as primer dimers. Therefore, H. pylori was not detected from any of the 290 oral samples. As a conclusion, H. pylori seems not to be permanently present in the oral cavity of a non-dyspeptic population.     

Prevalence of Streptococcus mutans with different colonial morphologies in human plaque and saliva

Abstract – Plaque and saliva samples were obtained twice from 58 children at an interval of 1 year and examined for the prevalence of Streptococcus mutans on Mitis salivarius (MS) and Mitis salivarius bacitracin (MSB) agar. Two types of S. mutans colonies with different colonial morphologies were seen on both media. They were serologically identified as serotypes c/e/f and d/g respectively. The first type, morphogroup c/e/f, had the typical "frosted-glass" appearance. It was the most prevalent and was found in 97% of the children. The second type, morphogroup d/g, had a creamy marzipan consistency with a dull, granular surface, gray to brown in color and often with some liquid around or on top of the colony. Group d/g was detected in 21 children (36%) and then together with colonies of group c/e/f. Children infected with single or multiple morphogroups of S. mutans generally harbored the same groups 1 year later. There was a significant positive correlation between the proportion of S. mutans in plaque and their numbers in saliva.     

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.     

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.     

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。     

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。     

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     

Effect of a fluoride-containing varnish on Streptococcus mutans in plaque and saliva

Abstract – The effect of topical application of a fluoride-containing varnish, Duraphat®, on the level of Streptococcus mutans in saliva and in dental plaque was investigated in schoolchildren. Samples of saliva and pooled buccal plaque were taken before varnish application and 4, 10 and 21 d after treatment. Fluoride varnish treatment with or without a preceding dental prophylaxis had no significant effect on the plaque and salivary levels of S. mutans. The findings suggest that the caries-reducing effect of fluoride varnish cannot be explained by an alteration of the incidence of 5. mutans in dental plaque or in saliva.     

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   

Molecular analysis of age‐related changes of Streptococcus anginosus group and Streptococcus mitis in saliva

The purpose of this study was to survey the prevalence of streptococcal species, especially Streptococcus anginosus (which has been reported to be associated with cancer in the upper digestive tract), Streptococcus constellatus, and Streptococcus intermedius in the saliva of different age groups. A sequence analysis of 16S rDNA was performed and DNA quantified using real‐time polymerase chain reaction. The S. anginosus level increased with age, whereas the levels of S. constellatus and S. intermedius did not change. Streptococcus mitis was the predominant species in the saliva of all the age groups but, unlike the S. anginosus, the proportion of S. mitis in the salivary bacteria decreased with age. The increase in S. anginosus with age should be carefully monitored because of its association with diseases, including cancer.     

不同龋敏感儿童菌斑中变异链球菌数量及菌群比例的定量分析

目的 比较不同龋敏感儿童口腔菌斑中变异链球菌数量及其在菌群中比例的差异。方法 采集26名3~4岁不同龋敏感的儿童牙面菌斑,运用TaqMan探针实时荧光定量聚合酶链反应（PCR）检测有龋组和无龋组儿童菌斑中变异链球菌和总菌数量,以及变异链球菌在总菌群中所占的比例,并对结果进行分析比较。结果 有龋组和无龋组儿童每毫克菌斑中变异链球菌菌落数分别为1.33×105、1.16×103 CFU·mg-1,二者间差异有统计学意义（P=0.033）;每毫克干重菌斑中总菌落数分别为7.17×107、1.01×108 CFU·mg-1,二者间差异无统计学意义（P=0.418）;有龋组和无龋组变异链球菌在总菌中所占比例分别为0.058 6和0.018 6,二者间差异有统计学意义（P=0.008）。结论 无龋与患龋儿童牙面总菌群数量差异无显著性,但患龋儿童牙面菌斑中变异链球菌数量更多,在总菌群中所占比例更大。提示菌斑中变异链球菌与总菌的比例与儿童患龋风险密切相关,可以作为评估龋易感性和预测龋病发展趋势的新指标。     

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。     

Adsorption of Streptococcus mutans lipoteichoic acid to hydroxyapatite

abstract – lipoteichoic acid extracted from cells of S. mutans strain BHT exhibited a high affinity for hydroxyapatite. Phosphate ions, fluoride ions and to a lesser extent human saliva inhibited or reversed this adsorption. Extracellular lipoteichoic acid preparations obtained from the supernatant of cultures of the same bacteria exhibited similar properties. It is suggested that lipoteichoic acids could play a significant role in the colonization of teeth by Gram-positive bacteria and thereby contribute to the formation and pathogenicity of dental plaque.     

Effect of SnF2, administered as mouthrinses or topically applied, on Streptococcus mutans, Streptococcus sanguis and lactobacilli in dental plaque and saliva

Abstract – Mouthrinsing with SnF₂ reduced the Streptococcus mutans population in plaque and saliva and the proportion of Streptococcus sanguis in plaque. The effect was of short duration: 2 weeks after treatment the values of S. mutans in plaque and saliva were even higher than the pretreatment values. Topical SnF₂ applications reduced the S. mutans population in plaque and saliva but did not reduce the proportion of S. sanguis in plaque. The eflect was more prolonged: 4 weeks after treatment the S. mutans population in interproximal plaque remained significantly reduced and the salivary levels of the organism had not fully returned to pretreatment levels. Both SnF₂ treatments significantly increased the salivary levels of lactobacilli. The values of laclobacilli in saliva remained signilicantly increased 4 weeks after the SnF₂ mouthrinsing but had almost returned to pretreatment levels within 2 weeks after the topical SnF₂ applications. The findings suggest that the cariogenic potential of dental plaque is differently affected depending on whether a drug is administered as a mouthrinse or is applied topically.