应用16 S rDNA基因芯片检测4种产黑色素感染根管可疑致病菌

目的:研制快速检测牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)、牙髓卟啉单胞菌(Porphyromonas endodontalis,Pe)、中间普氏菌(Prevotella intermedia,Pi)和变黑普氏菌(Prevotella nigrescens,Pn)的芯片系统.方法:利用Genebank中细菌16 S rDNA保守区序列,设计一对通用引物;通过已知Pg、Pe、Pi和Pn的16 S rDNA可变区序列,设计合成对应的特异性寡核苷酸探针.先用通用引物PCR扩增所有标准菌株的DNA并作荧光标记,PCR产物和已点样有Pg、Pe、Pi和Pn4种探针的芯片杂交,并用荧光扫描仪进行分析.结果:芯片上的Pg、Pe、Pi和Pn 4种探针只与相对应的Pg、Pe、Pi和Pn的PCR产物反应,而与其他标准菌株的PCR产物无反应.结论:应用16S rDNA基因芯片可以准确检测Pg、Pe、Pi和Pn,快速、灵敏、特异性高,有望在临床上得到应用.     

感染根管内产黑色素类杆菌的定植

目的:了解感染根管内产黑色素类杆菌(BPB)的定植情况.方法:采用16S rDNA PCR技术检测5种BPB在牙髓炎、慢性根尖周炎患牙根管内的定植情况.统计学分析5种BPB在感染根管内检出率的差异及菌种间相互关系.结果:BPB在感染根管内总检出率为60%,其中牙髓卟啉单胞菌(Pe)、变黑普氏菌(Pn)、牙龈卟啉单胞菌(Pg)、产黑普氏菌(Pm)、中间普氏菌(Pi)检出率分别为38%、32%、30%、18%、14%.Pe、Pn检出率明显高于Pm、Pi(P<0.05).Pe在慢性根尖周炎组检出率(50%)明显高于牙髓炎组检出率(20%)(P<0.05).Pn、Pg、Pi和Pm在慢性根尖周炎组检出率(43.33%、36.67%、16.67%、16.67%)与牙髓炎组检出率(20%、20%、10%、15%)无明显差异(P>0.05).Pg/Pe、Pg/Pn、Pe/Pn之间存在正相关.结论:BPB是感染根管内的优势菌群,是牙髓炎和慢性根尖周炎共有的致病菌.Pg/Pe、Pg/Pn、Pe/Pn常定植于同一根管.     

成人牙面色素沉积与口腔产黑菌关系的研究

目的:对口腔常见4类产黑菌进行检测,研究口腔产黑菌与口腔色素沉积的关系。方法:分别采集18～40岁牙面有色素沉积(非食用含色素食物和吸烟)和无色素沉积各30名受试者的牙菌斑样本,通过DNA抽提和二步PCR扩增方法,对口腔常见4类产黑菌:牙龈卟啉单胞菌(porphyromonas gingivalis,Pg)、牙髓卟啉单胞菌(porphyromonas endodontalis,Pe)、中间普氏菌(porphyromonas intermedius,Pi)、变黑普氏菌(Pre-votella nigrescens,Pn)进行检测。结果:所有样本均检测到目标细菌,30例无色素沉积者的标本中Pg、Pe、Pi、Pn的检出率分别为27%、40%、30%、87%;30例有色素沉积标本中的检出率分别为60%、80%、73.3%、80%。有色素沉积者的Pg、Pe、Pi的检出率明显高于无色素沉积者,差异有统计学意义(P<0.05);Pn二组无统计学差异(P﹥0.05)。结论:牙面有色素沉积者口腔产黑色素杆菌检出率高。     

牙龈卟啉菌rRNA基因间隔区的序列测定

目的通过rRNA基因间隔区碱基序列测定，建立对牙龈卟啉菌(Pg)菌株进行鉴定的技术。方法利用种特异性引物对Pg rRNA基因间隔区进行套式PCR扩增，用glass-milk纯化PCR扩增产物，对纯化的扩增产物进行碱基序列测定。结果琼脂糖凝胶电泳结果显示，Pg rRNA基因间隔区PCR扩增产物为0．88kb，经放射活性测序反应技术测得Pg rRNA基因间隔区的碱基序列。结论rRNA基因间隔区DNA扩增后进行测序，对Pg菌株鉴定是一种精确、重复性好的分子遗传学方法。     

AP-PCR法鉴别中间普里沃菌和变黑普里沃菌

目的　用引物OPA - 13的随机引物聚合酶链法 (arbitrarilyprimedpolymerasechainreaction ,AP -PCR)区分中间普里沃菌 (Prevotellaintermedia ,Pi)和变黑普里沃菌 (Prevotellanigrescens ,Pn)。 方法　用引物OPA - 13,采用AP -PCR法分析 97株来自 2 5例成年人牙周炎 (adultperiodontitis,AP)的Pi和 4 9株来自 2 1例AP的Pn的基因型。结果　 97株Pi中仅有 1株没有 90 0bp的特异性片段 ,4 9株Pn中也只有 1株没有产生 1.3kb的特异性片段。牙龈卟啉单胞菌 (Pg)ATCC 332 77,PgW 83,产黑色素普里沃氏菌ATCC 2 5 84 5 ,不解糖卟啉单胞菌ATCC 2 5 2 6 0 ,躯体普里沃氏菌ATCC 335 4 7,小齿普里沃氏菌ATCC 33185均不产生相关片段。结论　用引物OPA - 13的AP -PCR法可以区分Pi和Pn。     

产黑色素普氏菌DNA探针的制备及鉴定

本研究旨在应用DNA探针杂交技术快速检测产黑色素普氏菌、利用产黑色素普氏菌经酚抽取全染色体DNA，并以缺口转移法标记32P，制备探针。结果证实，该探针能够检测到膜上2×104的纯菌；当产黑色素普氏菌和中间普氏菌以不同比例混合点膜时，该探针能够检测到占混合菌群17％的目的菌；与口腔感染常见菌株杂交，未见交叉反应。     

实时荧光定量PCR法检测治疗后龈下菌斑中优势菌的变化

目的：运用实时荧光定量PCR(qRT-PCR)技术检测慢性牙周炎患者治疗前、后龈下菌斑中牙龈卟啉单胞菌(Pg)和中间普氏菌(Pi)的拷贝数,以了解qRT-PCR对2种牙周致病菌检测的敏感性和牙周治疗的疗效。方法：选取62例中、重度牙周炎患者,分别采用龈下刮治、牙周袋内盐酸米诺环素软膏(派丽奥)给药和两者综合治疗,采集治疗前、后(7d)龈下菌斑,提取细菌基因组DNA,合成针对Pg和Pi的16S rRNA基因的特异引物,运用qRT-PCR法检测Pg和Pi的拷贝数。采用SAS 9.1.3软件包对数据进行Kruskal-Wallis和 Wilcoxon 秩和检验。结果：Pg、Pi在不同样品组中检测到的绝对拷贝数数量分别为103～106和102～106,在慢性牙周炎患者龈下菌斑中的检出率和绝对拷贝数量显著高于健康对照组(P<0.05);3组治疗后的Pg数量比治疗前下降,其中综合治疗组和派丽奥治疗组下降率显著高于龈下刮治组。Pi的数量在派丽奥治疗组和龈下刮治组治疗后无显著减少,但在综合治疗组显著下降(P<0.05)。结论：qRT-PCR法能快速鉴定和精确定量Pg和Pi;综合治疗法比单一疗法能更有效抑制Pg和Pi。     

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。     

侵袭性牙周炎病原微生物的检测

目的检测侵袭性牙周炎（AgP）患者和牙周健康者龈下菌斑中的7种病原微生物,旨在寻找AgP的主要致病微生物.方法应用以16S rRNA为基础的聚合酶链反应（PCR）技术,检测55例AgP患者和17名健康对照者龈下菌斑中的7种牙周病原微生物：伴放线放线杆菌（Aa）,牙龈卟啉单胞菌（Pg）,福赛坦氏菌（Tf）,牙密螺旋体（Td）,直肠弯曲杆菌（Cr）,中间普氏菌（Pi）,变黑普氏菌（Pn）.结果55例AgP患者中仅有1例检测出Aa,而在健康对照者中未检出该菌.Pg、Tf、Td和Cr在AgP组的检出率分别为81.8%、83.6%、80.0%和81.8%,显著高于健康对照者（17.6%、11.8%、5.9%、29.4%）,差异有统计学意义（P＜0.01）.结论Pd、Tf、Td和Cr 4种微生物在AgP患者中有较高的检出率,提示它们的共同定植可能在AgP中起重要作用.     

基因芯片技术分析慢性根尖周炎患牙产黑色素菌

目的：利用基因芯片技术分析慢性根尖周炎患牙根管中牙髓卟啉单胞菌（Porphyromonas endodontalis,Pg）、牙龈卟啉单胞菌（Porphyromonas gingivalis,聊和中间普氏菌（Irevotella intermedia,Pi）三种产黑色素菌（blackpigmented bacteria,BPB）。方法：收集76例慢性根尖周炎患牙根管细菌学样本,经DNA抽提,PCR荧光标记后,与点样有忍、段和只三种菌特异寡核苷酸探针的基因芯片进行杂交,用激光共聚焦扫描仪分析杂交结果,最后进行统计分析。结果：慢性根尖周炎患牙中Pe、Pg和Pi三种菌的检出率分别为48．68％,32．89％,42．11％。Pe和Pg两菌同时被检出具有统计学意义（P〈0．05）。Pe和Pg与瘘管显著相关（p〈0．01）,Pe和Pg同时感染与瘘管、脓肿显著相关如〈0．01）。结论：产黑色素菌与慢性根尖周炎关系密切,Pe、Pg和瘘管、脓肿显著相关。用基因芯片技术分析慢性根尖周炎患牙中细菌具有快速、灵敏、高效的特点,而且可以通过增加基因芯片上探针数目扩展被检出菌的种类。     

应用聚合酶链式反应检测国人10种牙周病可疑致病菌

目的 :应用聚合酶链式反应分别检测国人不同牙周情况者龈下菌斑中 10种牙周可疑致病菌 ,观察其分布特点 ,并初步分析不同的细菌组合与牙周病的关系。方法 :从 3组对象 :健康组 ,龈炎组和牙周炎组中采取 12 4例龈下菌斑 ,提取DNA ,分别用 10种牙周病可疑致病菌的特异引物 ,采取聚合酶链式反应(PCR)扩增 16SrDNA片段来鉴定细菌种类。用统计软件分析细菌与各项临床指标的关系。结果 :经 χ2 检验 ,龈炎组和牙周病组牙龈卟啉菌的检出率高于健康组 (P <0 .0 5) ,牙周病组福塞氏类杆菌、中间型普里沃氏菌、齿垢密螺旋体 ,变黑普里沃氏菌的检出率高于健康组和龈炎组 (P <0 .0 5)。经Logistic回归进一步分析 ,中间型普氏沃氏菌、齿垢密螺旋体和变黑普里沃氏菌与牙周病关系更为密切 ;中间型普里沃氏菌、变黑普里沃氏菌和生痰二菌化碳噬纤维菌与附着丧失有关 ,而Pi和Td对探诊出血影响较大。结论 :我国人群的牙周病可疑致病菌的分布有自已的特点。     

Identification of periodontal pathogens in atheromatous plaques

BACKGROUND: Recent studies suggest that chronic infections including those associated with periodontitis increase the risk for coronary vascular disease (CVD) and stroke. We hypothesize that oral microorganisms including periodontal bacterial pathogens enter the blood stream during transient bacteremias where they may play a role in the development and progression of atherosclerosis leading to CVD. METHODS: To test this hypothesis, 50 human specimens obtained during carotid endarterectomy were examined for the presence of Chlamydia pneumoniae, human cytomegalovirus, and bacterial 16S ribosomal RNA using specific oligonucleotide primers in polymerase chain reaction (PCR) assays. Approximately 100 ng of chromosomal DNA was extracted from each specimen and then amplified using standard conditions (30 cycles of 30 seconds at 95 degrees C, 30 seconds at 55 degrees C, and 30 seconds at 72 degrees C). Bacterial 16S rDNA was amplified using 2 synthetic oligonucleotide primers specific for eubacteria. The PCR product generated with the eubacterial primers was transferred to a charged nylon membrane and probed with digoxigenin-labeled synthetic oligonucleotides specific for Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, and Prevotella intermedia. RESULTS: Eighty percent of the 50 endarterectomy specimens were positive in 1 or more of the PCR assays. Thirty-eight percent were positive for HCMV and 18% percent were positive for C. pneumoniae. PCR assays for bacterial 16S rDNA also indicated the presence of bacteria in 72% of the surgical specimens. Subsequent hybridization of the bacterial 16S rDNA positive specimens with species-specific oligonucleotide probes revealed that 44% of the 50 atheromas were positive for at least one of the target periodontal pathogens. Thirty percent of the surgical specimens were positive for B. forsythus, 26% were positive for P. gingivalis, 18% were positive for A. actinomycetemcomitans, and 14% were positive for P. intermedia. In the surgical specimens positive for periodontal pathogens, more than 1 species was most often detected. Thirteen (59%) of the 22 periodontal pathogen-positive surgical specimens were positive for 2 or more of the target species. CONCLUSIONS: Periodontal pathogens are present in atherosclerotic plaques where, like other infectious microorganisms such as C. pneumoniae, they may play a role in the development and progression of atherosclerosis leading to coronary vascular disease and other clinical sequelae.     

Relationship of periodontal bacteria and Porphyromonas gingivalis fimA variations with phenytoin-induced gingival overgrowth

OBJECTIVES: We investigated the relationship between phenytoin-induced gingival overgrowth (GO) and the harboring of periodontal bacteria. MATERIALS AND METHODS: Periodontal conditions and subgingival bacterial profiles were examined in 450 sites of 75 subjects. A polymerase chain reaction method was used to detect six bacterial species; Porphyromonas gingivalis (Pg), Actinobacillus actinomycetemcomitans (Aa), Tannerella forsythia, Treponema denticola (Td), Prevotella intermedia (Pi), and Prevotella nigrescens (Pn). Genetic variations of the Pg fimA gene were also examined. Bacterial occurrence was compared with the severity of GO, and alterations in the bacterial occurrence rate and quantities were monitored following periodontal treatment. RESULTS: The occurrences of Aa, Td, Pi, Pn, and Pg with type II fimA (type II Pg) were significantly associated with the severity of GO. Td occurrence was reduced in association with gingival improvement following ultrasonic scaling, however, no such relationship was observed with Aa, Pi, Pn, and Pg. In addition, Pg and Pi markedly persisted after treatment. Clinical improvement of the sites, following an Er:YAG laser treatment, significantly associated with quantitative reduction of Pg in improved sites, however, not that of Pi. CONCLUSION: Type II Pg and Td were each found to have a significant relationship with the development and deterioration of GO.     

寡发酵链球菌分子标识鉴定方法的建立

目的建立快速、有效鉴定寡发酵链球菌的方法，并验证其准确性。方法以9种口腔链球菌11株标准菌株的DNA为模板，用寡发酵链球菌16S rDNA的特异引物和乳酸氧化酶基因的引物通过PCR分别扩增这两个基因的部分片段，建立用分子标识鉴定该菌的方法。并从9个无龋的志愿者口腔中取得集合牙菌斑，在加红霉素的轻唾琼脂平板中进行培养，分离到疑似寡发酵链球菌的菌落后，用已建立的方法进行鉴定。并对初步鉴定为寡发酵链球菌的分离株进行16S rDNA序列同源性分析，以确定鉴定的准确性。结果用建立的分子标识鉴定方法，发现在11株标准菌株中只有3株寡发酵链球菌有扩增产物；用该方法鉴定为寡发酵链球菌的来自无龋志愿者牙菌斑的分离株，经16S rDNA序列同源性分析证实确实为寡发酵链球菌。结论用寡发酵链球菌16S rDNA特异性引物和乳酸氧化酶基因引物进行两步PCR来鉴定寡发酵链球菌是准确、可靠的。     

Pseudoramibacter alactolyticus in primary endodontic infections

A nested polymerase chain reaction (PCR)-based method was used to directly survey samples taken from primary endodontic infections for the occurrence of Pseudoramibacter alactolyticus. Identification by nested PCR was performed in root-canal samples from teeth associated with asymptomatic periradicular lesions or acute apical periodontitis, and in pus samples from acute periradicular abscesses. DNA was extracted from the samples and initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect a specific fragment of P. alactolyticus 16S rDNA. P. alactolyticus was detected in 76% of root-canal samples from teeth showing asymptomatic periradicular lesions, in 60% of samples taken from root canals associated with acute apical periodontitis, and in 32% of pus samples aspirated from acute periradicular abscesses. No significant association of this species with clinical symptoms was observed (p > 0.01). In general, P. alactolyticus occurred in 56% of samples taken from infections of endodontic origin. The high prevalence of P. alactolyticus in infections of endodontic origin as detected by nested PCR in this study, and its apparent pathogenicity, particularly in mixed infections, indicate that this bacterial species is a candidate endodontic pathogen that can participate in the etiology of different forms of periradicular diseases.     

Optimized oligonucleotides for the differentiation of Prevotella intermedia and Prevotella nigrescens

The gram-negative, anaerobic bacterium Prevotella intermedia plays an important role in the progression of periodontitis, whereas the etiological role of the closely related but phenotypically indistinguishable species Prevotella nigrescens is controversial. To differentiate between these species properly, 16S rDNA/RNA directed, computer-optimized oligonucleotides were designed and tested with 26 P. intermedia , 26 P. nigrescens and a number of closely and more distantly related strains. The oligonucleotides were used as primers in a polymerase chain reaction and could be demonstrated to be species specific with a detection limit of 50 bacterial cells, which could also be detected when diluted 1:1⁵ with different plaque bacteria. In addition, the described oligonucleotides were digoxigenin-labeled at the 3' end and used as DNA probes in a dot blot hybridization assay. This assay, although slightly less sensitive than the polymerase chain reaction-based method, gave species-specific reactions and also allowed, (semi-)quantification of bacterial cells in clinical specimens.     

Identification of Actinobacillus actinomycetemcomitans: polymerase chain reaction amplification of IktA–specific sequences

Actinobacillus actinomycetemcomitans has been strongly implicated in the etiology of localized juvenile periodontitis. Techniques used in the identification of this periodontal pathogen include cultural, biochemical, immunological and DNA hybridization analysis. In this study, we report the use of polymerase chain reaction (PCR) to amplify unique sequences of A. actinomycetemcomitans. Specific oligo-nucleotide primers LKT2 and LKT3 were designed to hybridize to the A. actinomycetemcomitans lktA gene, which encodes leukotoxin, a putative A. actinomycetemcomitans virulence factor. The LKT2 and LKT3 primers amplified lktA-specific sequences from all 12 A. actinomycetemcomitans strains tested. In another set of experiments, 13 other bacterial species, most of which are normal residents of the oral cavity, were tested with these primers. These PCR amplifications also contained 2 additional primers, RRN4 and RRN5, which served as positive controls; RRN4 and RRN5 were designed to amplify specific sequences of eubac-terial 16S ribosomal DNA (rDNA). PCR amplifications of all bacterial species tested, including A. actinomycetemcomitans , yielded 16S rDNA-specific DNA fragments. Furthermore, each bacterial species tested, with the exception of A. actinomycetemcomitans , failed to amplify lktA sequences. The LKT and RRN primers were used in further PCR experiments to detect A. actinomycetemcomitans directly from gingival fluid samples. The results clearly demonstrate the simplicity, rapidity, specificity and accuracy of the LKT primers in the identification of A. actinomycetemcomitans.     

Novel subgingival bacterial phylotypes detected using multiple universal polymerase chain reaction primer sets

INTRODUCTION: Molecular ecological analysis based on 16S rRNA gene sequence analysis is well established for the characterisation of complex bacterial communities. However, 'universal' PCR primers can introduce biases into the analysis of the species composition of clone libraries because of mismatches between the primer and target organism sequences. In this study, three universal primer sets were compared for the analysis of the microflora in subgingival plaque. METHODS: Three subgingival plaque samples were collected from two subjects with localised severe chronic periodontitis. Half of each sample was cultured while DNA was extracted from the remaining half and 16S rDNA amplified with universal primer pairs 27F, 1525R (A); 27F, 1492R (B) and 530F, 1525R (C). Amplified genes were cloned, sequenced and identified by comparison with 16S rRNA databases. RESULTS: 137 taxa were identified among 177 isolates and 417 clones sequenced. Of these, 86 were detected only by the molecular technique whereas 26 were found only by culture. Sequences from 81 taxa did not correspond to those of named species and of these, 38 were not represented in the nucleotide databases. 16S RNA genes for these 38 taxa were sequenced and deposited with GenBank. CONCLUSION: The use of three sets of universal primers allowed the identification of 38 novel bacterial phylotypes. There were marked differences in the composition of the libraries generated by the different primer sets. A combination of molecular and cultural techniques is recommended to maximise the coverage of detection of bacterial taxa in oral samples.