反向基因芯片技术检测牙龈卟啉单胞菌致病岛基因

目的：应用反向基因芯片技术检测PG0836、PG0838、PG0839基因在慢性牙周炎患者和牙周健康者牙龈卟啉单胞菌（P.gingivalis）中分布,探讨这些基因与牙周临床指数之间的关系。方法：选取41例慢性牙周炎患者,牙周健康者76例,记录临床指标牙周探诊深度、临床附着丧失、探诊出血及牙齿松动度,取龈下菌斑进行细菌分离培养,以临床采集的样本提取的DNA为探针,以抑制消减杂交技术获得的P.gingivalis W83的特异基因片段PG0836、PG0838、PG0839为目标序列,采用Cy5荧光标记目标序列。应用基因芯片技术检测PG0836、PG0838、PG0839基因在病变部位及非病变部位的牙龈卟啉单胞菌中的分布。结果：PG0836、PG0838、PG0839基因在病变部位和非病变部位中的检出率均有统计学差异（P〈0.05）,并且与牙周临床指数PD、CAL、TM相关。结论：PG0836、PG0838、PG0839基因与P.gingivalis的致病性有关。     

牙龈卟啉单胞菌PG0717基因与慢性牙周炎的相关性研究

目的检测慢性牙周炎患者和牙周健康者龈下菌斑中牙龈卟啉单胞菌（P.gingivalis）PG0717基因,探讨PG0717基因与牙周临床指数之间的关系。方法选取慢性牙周炎（CP）患者90例和牙周健康者90例,共采集龈下菌斑标本540个;记录临床牙周指数（牙周探诊深度、临床附着丧失和探诊出血）;设计特异性引物检测P.gingivalis阳性龈下菌斑标本的PG0717基因。结果在P.gingivalis阳性龈下菌斑中,CP组PG0717基因检出率显著高于对照组,分别为56.22%和41.27%（掊2=4.50,P＜0.05）;随着牙周探诊深度、临床附着丧失加重和探诊出血趋势的增加,CP组该基因检出率呈现增高趋势。结论PG0717基因与P.gingivalis的致病性有关。     

牙龈卟啉单胞菌与牙周病相关性的聚合酶链反应研究

目的利用PCR检测慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌(Porphyromonas gingivalis,P. g)的16S rDNA水平,通过检测该基因水平来探讨牙龈卟啉单胞菌的水平与牙周病的相关性.方法采集慢性牙周炎患者12例共36个龈下菌斑标本,记录临床指标(探诊深度、临床附着水平、牙龈指数、菌斑指数、龈沟出血指数),PCR检测龈下菌斑标本中的P. g 16S rDNA基因,扩增产物经琼脂糖电泳、拍照后,应用计算机软件GeneTools扫描并计量其中的相对核酸含量.结果P. g16S rDNA水平与探诊深度(P＜0.001)及牙龈指数(P＜0.01)之间存在正相关关系.结论P.g16S rDNA水平与牙周状态密切相关,对于P.g16S rDNA水平的监测有望成为牙周病诊断和治疗方案制定的辅助检查手段.     

牙龈卟啉单胞菌毒力岛基因检出与慢性牙周炎的相关性研究

目的:检测慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌(Porphyromonas gingivalis)毒力岛中PG0836、PG0838和PG0839基因,探讨3个基因与临床牙周指数之间的关系.方法:选取慢性牙周炎(chronic periodoiltitis,CP)患者90例,共采集龈下菌斑标本270个.记录受试位点的牙周探诊深度、临床附着丧失和探诊出血情况.设计特异性引物,检测P.gingivalis阳性龈下菌斑标本的PG0836、PG0838和PG0839基因.采用SPSS 11.0软件包进行统计学分析,采用X2检验对不同牙周临床指数基因的检出率进行比较.结果:在CP患者P.gingivalis阳性龈下菌斑中,PG0836、PG0838和PG0839基因检出率分别为66.17%、24.88%和27.86%.探诊出血阳性位点的PG0839基因检出率(28.35%)显著高于探诊出血阴性位N(14.29%,P<0.05).在牙周探诊深度为4～6mm和>6mm的受试位点,PG0839基因检出率均显著高于牙周探诊深度<4mm的位点(P<0.05).PG0836和PG0838基因在不同牙周指数组检出率无显著差异.结论:PG0839基因的检出与CP病损部位的临床指标呈正相关关系,提示该基因可能与P.gingivalis的致病性有关.     

青春期龈炎龈下菌斑中牙龈卟啉单胞菌kgp基因型的研究

目的 研究青春期龈炎龈下菌斑中牙龈卟啉单胞菌（P.gingivalis）的特异kgp基因型，评估其与疾病严重程度之间的关系。方法检查并记录青春期龈炎组与牙龈健康组各36例的牙周临床指数，收集龈下菌斑样本，提取染色体DNA，采用聚合酶链反应（PCR）方法扩增编码牙龈蛋白酶K（KGP）催化域的序列片段。PCR产物用限制性内切酶Mse I消化。结果 P.gingivalis有毒株W83表现为kgp-A型，而无毒株ATCC33277表现为kgp-B型。所有P.gingivalis阳性个体的龈下菌斑中只检测到一种kgp基因型。kgp-A型在青春期龈炎组P．gingivalis阳性个体中的检出率为79．0％，在牙龈健康组为22．2％，两组kgp基因型的检出率差异有统计学意义（P=0．028）；青春期龈炎组有P.gingivalis定植的个体中，牙周临床指数与kgp的基因型无相关关系（P〉0．05）。结论青春期龈炎个体定植的Pgingivalis的kgp基因型多与有毒株P.gingivalis W83的表现相同，有必要监测kgp-A型阳性的个体，因其个体发展为牙周疾病的危险度可能会增高。     

牙龈卟啉单胞菌脂蛋白基因在慢性牙周炎及牙周健康者中的检测

目的 应用基因芯片技术检测3种脂蛋白基因PG0717、PG0183、PG2135在慢性牙周炎患者和牙周健康者牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)中的分布,探讨这些基因与牙周临床指数之间的关系,为研究脂蛋白在Pg致病过程中的作用提供依据.方法 选取41例慢性牙周炎患者(牙周炎组)及76例牙周健康者(健康对照组),记录探诊深度、附着丧失、探诊出血及牙齿松动度,取龈下菌斑进行细菌分离培养,以临床采集的样本提取的DNA为探针,以抑制消减杂交技术获得的PgW83特异基因片段PG0717、PG0183、PG2135为目标序列,采用Cy5荧光标记目标序列.应用基因芯片技术检测PG0717、PG0183、PG2135基因在牙周炎组病变部位、非病变部位和健康对照组Pg中的分布.结果在牙周炎组病变部位PG0717、PG0183、PG2135基因的检出率分别为90%(18/20)、70%(14/20)、70%(14/20),非病变部位的检出率分别为60%(12/20)、45%(9/20)、40%(8/20),而在健康对照组PG0717、PG0183、PG2135的检出率分别为55%(11/20)、25%(5/20)、30%(6/20).3种脂蛋白基因在牙周炎组病变部位和健康对照组中的检出率差异均有统计学意义(P＜0.05),并且与牙周探诊深度、临床附着丧失、探诊出血及牙齿松动度相关.结论 带有PG0717、PG0183、PG2135基因的Pg菌株致病力强.     

成人牙周健康状况与fimA基因型牙龈卟啉单胞菌的相关性

目的分析不同fimA基因型牙龈卟啉单胞菌（P.gingivalis）在牙周健康人群和慢性牙周炎人群中的分布,探讨不同fimA基因型P.gingivalis与成人牙周状况的相关关系。方法收集牙周健康组（136例）和慢性牙周炎组（115例）的龈下菌斑样本,采用16S rRNA PCR法检测P.gingivalis,并根据各fimA基因型（Ⅰ～Ⅴ和Ⅰb）的特异性引物检测不同fimA基因型P.gingivalis菌株的分布,计算OR值和95%可信区间。结果牙周健康组和慢性牙周炎组龈下菌斑样本中P.gingivalis阳性率分别为22.1%和81.7%,多数样本中只检测到1种fimA基因型。牙周健康组中ⅠfimA型的检出率最高（占66.7%）;慢性牙周炎组中则为ⅡfimA基因型（占43.6%）,其次为Ⅳ和Ⅰb fimA基因型。慢性牙周炎的发生与P.gingivalis的关系密切（OR=16.36）,Ⅰ、Ⅰb、Ⅱ、Ⅲ、Ⅳ、ⅤfimA基因型P.gingivalis与慢性牙周炎相关性的OR值分别为0.97、13.26、36.62、4.57、22.86、1.19;ⅡfimA基因型P.gingivalis与慢性牙周炎的相关性最强,其次为Ⅳ和Ⅰb型。结论P.gingivalis菌株的fimA基因型存在差异,特异性fimA基因型P.gingivalis可能与成人慢性牙周炎的发生关系密切。     

龈沟液中有机酸与龈下牙龈卟啉单胞菌间的关系

目的分析慢性牙周炎患者相同龈沟液样本中挥发性有机酸与牙龈卟啉单胞菌（Porphyromonas gingivalis,P.g）间的关系。方法应用高效毛细管电泳技术分析慢性牙周炎患者治疗前龈沟液中有机酸的浓度;应用多聚酶链反应（polymerase chain reaction,PCR）技术检测相同样本中牙龈卟啉单胞菌。结果慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌检出率显著高于牙周健康对照者;慢性牙周炎患者P.g阳性组龈沟液中丁酸与异戊酸浓度显著高于P.g阴性组。结论结果表明相同样本龈沟液中丁酸与异戊酸的表达与P.g的检出相关联,P.g是重要牙周致病菌     

不同fimA基因型牙龈卟啉单胞菌在牙周健康人群中的分布

目的调查不同fimA基因型牙龈卟啉单胞菌 (P.gingivalis)在牙周健康人群中的分布情况。方法收集136例牙周健康者的龈下菌斑样本,采用16S rRNAPCR法检测P.gingivalis;并根据各fimA基因型特异性引物,用PCR法检测不同fimA基因型P.gingivalis的分布。 结果136例牙周健康者的龈下菌斑样本中携带P.gingivalis的阳性率为22.1%。大多数受检者龈下菌斑中只检测到1种fimA基因型 P.gingivalis菌株(80.0%);5例样本检出了2种fimA型P.gingivalis(16.7%),且均为ⅠfimA型与其它fimA 型P.gingivalis的联合检出。各fimA型P.gingivalis的检出情况:Ⅰ型(66.7%)、Ⅰb型(6.7%)、Ⅱ型(6.7%)、 Ⅲ型(10%)、Ⅳ型(6.7%)、Ⅴ型(16.7%)。Ⅰ型的检出率明显高于其它各型,差异有统计学意义(P<0.05);其它各fimA型 P.gingi-valis间的检出差异均无统计学意义。结论本研究条件下ⅠfimA型P.gingivalis在牙周健康人群中检出例数最高,提示:我 国牙周状况不同人群携带的P.gingivalis菌株fimA基因型可能存在差异。?     

龈下菌斑中牙龈卟啉单胞菌牙龈素基因片段的检测

目的：检测慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌（Porphyromonas gingivalis,P.gingivalis）的2个基因片段kgp-cd和rgpB-cd,探讨2个基因片段的存在和缺失与牙周临床指标之间的关系.方法：选择慢性牙周炎患者龈下菌斑84个,对P.gingivalis阳性的龈下菌斑样本进行kgp-cd和rgpB-cd基因片段检测;根据2个基因片段的有无,将P.gingivalis分为A型和B型,采用SPSS11.5统计软件包,用t检验和x2检验分析不同基因型P.gingivalis与牙周临床指标的关系.结果：A型P.gingivalis在慢性牙周炎中的检出率高于B型（P＜0.05）,分别为85.29%和14.71%.不同基因型P.gingivalis引起的牙周袋探诊深度和牙龈出血倾向存在显著性差异（PD：t=2.85,P＜0.05;BOP：P＜0.05）.结论：kgp-cd和rgpB-cd基因与P.gingivalis的致病性有关.     

牙龈卟啉单胞菌在龈下菌斑和颊黏膜中的检测

目的　检测牙周健康者及牙周炎患者在颊黏膜和龈下菌斑中牙龈卟啉单胞菌的阳性率,探讨其与牙周炎发生和发展的关系。方法　选取40例牙周健康者和39例慢性牙周炎患者,分别收集颊黏膜和龈下菌斑样本,提取细菌DNA,设计细菌通用引物和牙龈卟啉单胞菌的特异引物用于PCR扩增,检测牙龈卟啉单胞菌的阳性率。结果　牙周健康组菌斑样本和颊黏膜样本牙龈卟啉单胞菌的阳性率分别为37·5%和32·5%,而牙周炎组菌斑样本和颊黏膜样本牙龈卟啉单胞菌的阳性率分别为69·23%和46·15%。牙周炎组菌斑的牙龈卟啉单胞菌阳性率高于牙周健康组,颊黏膜的牙龈卟啉单胞菌阳性率在组间无统计学差异;牙周炎组菌斑牙龈卟啉单胞菌阳性率高于颊黏膜, 牙周健康组两部位阳性率无统计学差异。结论　牙龈卟啉单胞菌除在菌斑中有高检出率外,在颊黏膜中也有较高的检出率,提示颊黏膜也是牙周细菌在口腔定植的重要部位,牙龈卟啉单胞菌也可在健康人群中检出,提示其有可能是口腔内固有菌群之一。     

Evaluation of a non-radioactive DMA probe for detecting Porphyromonas gingivalis in subgingival specimens

This study compared the ability of a nonradioactive digoxigenin-labeled DNA probe and anaerobic culture to identify subgingival Porphyromonas gingivalis. Total cellular DNA from P. gingivalis ATCC 33277T was labeled using the Genius kit from Boehringer Mannheim Biochemicals. Anaerobic culture was performed using VMGA III transport medium and enriched brucella blood agar. The DNA probe could detect as little as 1000 P. gingivalis cells added to supragingival plaque. Also, the probe could detect P. gingivalis when it was present in proportions too low to be visualized on overgrown bacterial plates. The probe showed no visible reaction with strains of various oral species or with thousands of non-P. gingivalis colonies from plaque samples. VMGA III could maintain the viability of P. gingivalis for up to 6 days, as evidenced by DNA probing of colony blot of subgingival cultures. A total cellular DNA probe for detecting P. gingivalis seems to offer a simple and reliable method of detecting the organism in subgingival specimens.     

慢性牙周炎患者龈下菌斑中不同基因型牙龈卟啉单胞菌的检测

摘要 目的:建立临床标本中牙龈卟啉单胞菌(P.g)的PCR检测方法,探讨慢性牙周炎患者不同牙位的龈下菌斑中P.g基因型的差异。方法:采用培养法分离鉴定慢性牙周炎患者不同牙位龈下菌斑中P.g,同时采用PCR检测 P.g16SrDNA、prtC和fimA基因。部分扩增产物测定了核苷酸序列。结果:在66例患者的127个龈下菌斑标本中, P.g16SrDNA、prtC和fimA多重引物扩增的阳性率为9814%;PCR阳性率显著高于培养法P.g的检出率(P< 0101)。3010%的患者(18/60)同时感染了不同基因型的P.g菌株。P.g16SrDNA、prtC和fimA扩增片段的核苷酸序列同源性在98162%~100%之间。结论:本文所建立的P.g的PCR检测方法具有较高的敏感性和特异性,适用于P.g的快速临床诊断。同一患者可被不同感染来源的多株P.g同时感染。     

Porphyromonas gingivalis lipids and diseased dental tissues

BACKGROUND/AIM: Porphyromonas gingivalis synthesizes several classes of dihydroceramides and at least one of these lipid classes promotes proinflammatory secretory reactions in gingival fibroblasts as well as alters fibroblast morphology in culture. The purpose of this investigation was to determine whether the dihydroceramide lipids of P. gingivalis are recovered in lipid extracts of subgingival plaque, diseased teeth, and diseased gingival tissue samples. METHODS: Lipids were extracted from P. gingivalis, subgingival plaque, subgingival calculus, teeth laden with gross accumulations of subgingival calculus, and gingival tissue samples obtained from chronic severe periodontitis sites. Lipid samples were analyzed by gas chromatography-mass spectrometry as trimethylsilyl derivatives or by electrospray-mass spectrometry as underivatized products. High-performance liquid chromatography fractions of P. gingivalis lipids and gingival tissue lipids were also analyzed by electrospray-mass spectrometry analysis. RESULTS: P. gingivalis phosphorylated dihydroceramides were recovered in lipid extracts of subgingival plaque, subgingival calculus, calculus contaminated teeth, and diseased gingival tissue samples. However, the distribution of phosphorylated dihydroceramides varied between these samples. CONCLUSION: Subgingival plaque, subgingival calculus, diseased teeth, and gingival tissue are contaminated with phosphorylated dihydroceramides produced by P. gingivalis. The previously reported biological activity of these substances together with the recovery of these lipids at periodontal disease sites argues strongly for their classification as virulence factors in promoting chronic inflammatory periodontal disease.     

Detection frequency of periodontitis-associated bacteria by polymerase chain reaction in subgingival and supragingival plaque of periodontitis and healthy subjects

The aim of this study was to compare the detection frequencies of 25 bacterial species in subgingival and supragingival plaque of 18 untreated periodontitis subjects and 12 periodontally healthy subjects. Genomic DNA was extracted from subgingival and supragingival plaque samples, and bacterial detection was performed by polymerase chain reaction of the 16S rRNA genes. Fourteen bacteria showed no relationship with periodontitis, and 11 of these 14 species were frequently detected (≥50%) in subgingival plaque in both periodontitis and healthy subjects. Nine bacteria such as Eubacterium saphenum, Prevotella intermedia, and Treponema denticola seemed to be related to periodontitis; their detection frequencies in subgingival plaque samples were higher in periodontitis than in healthy subjects, but these differences were not statistically significant by multiple comparisons (0.002≤ P< 0.05). Two species ( Mogibacterium timidum and Porphyromonas gingivalis ) were detected significantly more frequently in subgingival plaque of periodontitis subjects than of healthy subjects ( P< 0.002), with P. gingivalis being detected only in periodontitis subjects, suggesting that these two species are closely related to periodontitis. There were no significant differences in the detection frequencies of the 25 bacteria between subgingival and supragingival plaque, suggesting that the bacterial flora of supragingival plaque reflects that of subgingival plaque.     

Comparison of the microbiota of supra- and subgingival plaque in health and periodontitis

BACKGROUND, AIMS: The purpose of the present investigation was to compare the microbial composition of supra and subgingival plaque in 22 periodontally healthy (mean age 32+/-16 years) and 23 adult periodontitis subjects (mean age 51+/-14 years). METHODS: A total of 2358 supra and separately subgingival plaque samples were collected from the mesial aspect of all teeth excluding 3rd molars in each subject. Samples were examined for the presence and levels of 40 bacterial taxa using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Clinical assessments including dichotomous measures of gingival redness, bleeding on probing, plaque accumulation and suppuration, as well as duplicate measures of pocket depth and attachment level, were made at 6 sites per tooth. Mean counts (x10(5), % DNA probe count and % sites colonized for each species were determined separately for supra and subgingival samples in each subject and then averaged across subjects in the 2 clinical groups. Significance of differences between healthy and periodontitis subjects was determined using the Mann-Whitney test and adjusted for multiple comparisons. RESULTS: Mean total DNA probe counts (x10(5), +/-SEM) for healthy and periodontitis subjects in supragingival plaque were 72.1+/-11 and 132+/-17.5, respectively (p<0.01), and in subgingival plaque 22.1+/-6.6 and 100.3+/-18.4, (p<0.001). Porphyromonas gingivalis, Bacteroides forsythus and Treponema denticola could be detected in supragingival plaque samples of both healthy and periodontitis subjects. Actinomyces species were the dominant taxa in both supra- and subgingival plaque from healthy and periodontitis subjects. 4 Actinomyces species accounted for 63.2%, of supragingival and 47.2% of subgingival plaque in healthy subjects and 48.% and 37.8% in periodontitis subjects respectively. Increased proportions of P. gingivalis, B. forsythus, and species of Prevotella, Fusobacterium, Campylobacter and Treponema were detected subgingivally in the periodontitis subjects. P. gingivalis, B. forsythus and T. denticola were significantly more prevalent in both supra- and subgingival plaque samples from periodontitis subjects. CONCLUSIONS: The main differences between supra and subgingival plaque as well as between health and disease were in the proportions and to some extent levels of Actinomyces, "orange" and "red" complex species.     

Microbiologic analysis of periodontal pockets and carotid atheromatous plaques in advanced chronic periodontitis patients

BACKGROUND: In recent years, many researchers have focused their attention on the ability of periodontal pathogens to colonize atheromatous plaques. Nevertheless, a clear correlation between the detection rates of periodontopathic bacterial DNA in atheromas and in subgingival plaque samples has not been established. The aim of our study was to assess the presence of five periodontal pathogens (Actinobacillus actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia [formerly Tannerella forsythensis]) in periodontal pockets and in carotid atheromas recovered from the same patients. METHODS: Thirty-three patients with advanced chronic periodontitis scheduled for endarterectomy were enrolled in the study. DNA was extracted from subgingival plaque samples and carotid atheromas. Universal bacteria primers for general detection of bacteria and species-specific primers for detection of periodontal pathogens were used to amplify part of the 16S rRNA gene by polymerase chain reaction. RESULTS: All subgingival plaque samples were positive for at least one target microorganism. The prevalence of T. forsythia, P. gingivalis, T. denticola, P. intermedia, and A. actinomycetemcomitans were 69.7%, 63.6%, 54.5%, 45.4%, and 33.3%, respectively. Bacterial DNA was detected in 31 out of 33 endarterectomy specimens. However, none of the samples tested positive for DNA from periodontal pathogens. CONCLUSION: The presence of periodontal bacteria in atheromatous plaques was not confirmed by this investigation; thus, no correlation could be drawn between periodontitis bacteria and microorganisms involved in the atherosclerotic lesions.