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16S rDNA结合膜芯片检测3种产黑色素牙周可疑致病菌

目的:探讨采用16SrDNA结合膜芯片检测牙龈卟啉菌Pg、中间普氏菌Pi和变黑普氏菌Pn的价值。方法:利用Genebank中细菌16SrDNA保守区序列,设计一对通用引物,通过已知Pg、Pi和Pn的16SrDNA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,PCR产物和已点样有Pg、Pi和Pn的3种探针的膜芯片杂交,进行分析。结果:膜芯片上的Pg、Pi和Pn3种探针只能与相对应的Pg、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:用16SrDNA结合膜芯片,可准确检测Pg、Pi和Pn,具有很高的特异性,有望成为一种有效的临床检测方法。     

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.     

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。     

基因检测技术在龈下微生物鉴定中的新进展

近年来,非放射性载体分子标记在龈下菌斑微生物鉴定中被广泛应用,它是一种快速的、具有高度敏感性和特异性的检测手段。本文对DNA的提取和纯化,DNA探针的标记作了介绍。反向杂交是将未知菌作探针,与纯化的参考菌株DNAs杂交技术;rRNA基因探针可检测同源性很高的细菌;多聚酶链反应是在体外用寡核苷酸引物及Taq酶合成特异核苷酸序列的方法,也广泛用于各个领域。     

牙龈卟啉菌和牙垢密螺旋体在不同深度牙周袋内的分布

目的:建立检测牙龈卟啉菌(Porphyromonas gingivalis,Pg)和牙垢密螺旋体(Treponema denti-cola,Td)的实时荧光定量PCR方法,探讨Pg和Td的定植量与牙周状况的关系.方法:建立SYBRGreenⅠ荧光实时定量PCR法检测Pg和Td细菌数量的方法.采集43例慢性牙周炎患者的龈下菌斑并检测Pg和Td的检出率和细菌量,同时记录取样位点的牙周探诊深度.结果:在102～107范围内细菌数与Ct值有良好的线性关系,最低检测到Pg和Td的DNA模板为100拷贝.Pg和Td在慢性牙周炎患者中的阳性检出率、共同检出率和两种细菌数量均随着牙周探诊深度的增加而升高,多组探诊深度间有统计学差异.结论:慢性牙周炎的牙周状况与Pg和Td的细菌数量密切相关.     

牙周病和龋病致病菌的共聚及共聚抑制

目的 :研究牙周病和龋病致病菌的共聚和共聚抑制关系及特征 ,筛选有效共聚抑制剂。方法 :以 4株牙周致病菌 (具核梭杆菌、伴放线放线杆菌、牙龈卟啉单胞菌和中间普氏菌 )和 4株致龋菌 (变形链球菌、血链球菌、粘性放线菌和嗜酸乳杆菌 )的国际标准菌株为研究对象 ,通过目测法和比浊法确定各组合细菌的共聚度并研究乳糖和精氨酸对共聚度 > 2 (或 >2 0 % )共聚对的共聚抑制作用。结果 :Fn Av、Pg Av、Fn Sm、Fn Ss、Fn La和Pg Ss共聚度 >2 0 % ;4.5～ 6.0mM精氨酸对除Fn Av外 5对有明显抑制作用 ,解聚度为 49%～ 92 % ;12 0～ 3 0 0mM乳糖仅对上述 6对共聚对中的Pg Ss有明显抑制作用 ,解聚度为 5 7%～ 91% ,对Pg Av有部分抑制作用 ,最大解聚度约为 3 5 % ,但对Fn G 菌几乎无效。结论 :牙周致病菌和致龋菌之间存在高度特异的共聚反应 ,精氨酸为有效共聚抑制剂。     

嗜酸乳杆菌及其灭活菌粘附及拮抗牙周致病菌特性研究

目的研究体外共培养时,嗜酸乳杆菌（lactobacillus acidophilus,La）活菌及其灭活菌对牙龈角化上皮细胞株KB细胞的粘附,以及La对口腔致病菌具核梭杆菌（fusobacteriurn nuclearum,Fn）和牙龈卟啉单胞菌（porphyromonas gingivalis,Pg）的粘附拮抗作用,以期为牙周疾病益生菌防治提供实验依据。方法选取不同生命状态（活菌、灭活菌）的La4356作为研究菌,运用光镜以及革兰染色法,分析不同状态的嗜酸乳杆菌对口腔牙龈上皮细胞株KB细胞的粘附指数,以及La作用于Pg和Fn后,Pg和Fn粘附指数的变化。结果两种生命状态的嗜酸乳杆菌对KB细胞均有粘附作用,活菌的粘附指数略高于灭活菌。La和其灭活菌均可与Pg和Fn竞争对KB细胞的粘附,显著降低Pg和Fn对KB细胞的粘附指数;同时在允许浓度范围内,高浓度的益生菌的粘附拮抗作用较低浓度时明显。当益生菌和致病菌浓度比大于1：1时,La灭活菌抑菌作用略强于La活菌。结论益生菌La及其灭活菌均可粘附于上皮细胞,且均对Pg、Fn具有较强的粘附拮抗作用,可用作牙周生态防治。     

伴放线放线杆菌全染色体DNA探针的制备和鉴定

本研究的目的是制备伴放线放线杆菌全染色DNA探针,并评价其特异性和敏感性。抽提标准菌株Aa Y4 DNA,经~(32)P标记后制成探针,然后用斑点杂交法鉴定探针性质。结果Aa全染色体DNA探针的灵敏性为8×10~2纯菌或1ng同源DNA,探针与口腔常见菌的杂交显示仅与嗜沫嗜血杆菌有较弱的交叉反应。提示~(32)P标记Aa全染色体DNA探针的敏感性、特异性均较高,且方便实用,有临床推广应用价值。     

变形链球菌表面抗原Ⅰ／Ⅱ基因体外扩增片段的Southern杂交…

研究扩增产物的特异性，将变链菌ＮＧ８ｓｐａＰ基因１０１７ｂｐ扩增产物，采用随机引物标记法，使用α－＾３２ｐｄＣＴＰ标记成ＤＮＡ探针，与１３株变链菌相应的扩增产物进行Ｓｏｕｔｈｅｒｎ杂交。６株有强杂交信号，与ＰＣＲ结果一致，说明扩增产物是特异的。     

牙龈卟啉菌特异基因片段的体外扩增和鉴定

Ｐｇ与口腔感染性疾病关系密切．现根据Ｐｇ特异的菌毛亚单位蛋白结构基因设计寡聚核苷酸引物，采用ＰＣＲ技术扩增了其中长５４２ｂｐ的片段并对其进行了特异性鉴定。结果表明：所设计的引物能从５株Ｐｇ菌中扩增出大小一致的特异ＤＮＡ片段，但不能从其它１８株异种菌中扩增出产物，故具有高度的种特异性．其检测的敏感性为０．ｌｎｇ，为ＰＣＲ应用于口腔致病菌的检测奠定了基础。     

牙龈卟啉菌特异DNA片段的分子克隆

采用ＰＣＲ扩增Ｐｇ特异ＤＮＡ片段作为外源目的ＤＮＡ，将质粒载体ｐＵＣ１９和外源ＤＮＡ用限制性内切酶ＰｓｔＩ和ＢａｍＨＩ消化，经过连接形成重组质粒并转化细菌后，选白色菌落进行鉴定。结果证实我们成功地获得了含Ｐｇ特异基因片段的克隆。通过将细菌增菌后就可得到取之不尽的特异ＤＮＡ片段。为研究Ｐｇ的基因结构，致病机理以及制备特异的核酸探针检测细菌奠定了基础。     

3种寡核苷酸探针对龈下菌斑中牙周致病菌的检测

目的　采用寡核苷酸探针研究龈下菌斑中3种牙周致病菌的分布。方法　利用3种寡核苷酸探针对 60例慢性牙周炎患者60个患病位点、10例健康人的10个健康对照位点龈下菌斑中的牙龈卟啉单胞菌、福赛类杆菌、牙密螺旋体进行检测。结果　牙周炎位点龈下菌斑中的牙龈卟啉单胞菌、福赛类杆菌、牙密螺旋体的检出率分别为91·67%,90·00%和95·67%,明显高于健康对照位点;有83·33%的牙周炎位点同时检出3种致病菌,3种细菌检出情况为两两正相关(P<0·01)。结论　牙龈卟啉单胞菌、福赛类杆菌、牙密螺旋体在慢性牙周炎患者龈下菌斑中的检出率很高,它们间可能存在相互协同致病作用。     

Species-specific DNA probe for the detection of Porphyromonas gingivalis from adult Chinese periodontal patients and healthy subjects

BACKGROUND: It has been reported that Porphyromonas gingivalis is closely associated with chronic periodontitis and its detection has been recommended as a routine marker for periodontal diagnosis. The purpose of this study was to evaluate the sensitivity and specificity of a DNA probe to detect P. gingivalis in adult Chinese periodontitis patients as well as to find a rapid and convenient method to detect P. gingivalis in clinical practice. METHODS: A total of 26 bacterial strains (20 reference strains and 6 clinical isolates) were collected, of which 5 were P. gingivalis and 21 were heterologous species. A DNA fragment of 542 bp, which encodes the fimbriae subunit protein (fimA) of P. gingivalis, was obtained by polymerase chain reaction (PCR) and molecular cloning techniques and used to construct the DNA probe, labeled with 32P or with digoxigenin. The constructed DNA probe was used to detect P. gingivalis in specimens collected from the periodontal pockets of 100 patients clinically confirmed with chronic periodontitis. One hundred periodontally healthy persons served as a control group. RESULTS: Positive reactions were seen in all 5 strains of P. gingivalis while no visible reaction was found in other species. The DNA probe was capable of detecting as few as 100 P. gingivalis cells in samples. A significant difference in the positive rates of P. gingivalis between the periodontitis patients and the control group was found (P<0.01). In addition, the amount of detected P. gingivalis was positively correlated with the extent of tooth mobility, depth of periodontal pockets, and patient age (P<0.01). CONCLUSION: The DNA probe is specific and sensitive for the detection of P. gingivalis in chronic periodontitis specimens and may be a suitable method for the clinical diagnosis of chronic periodontitis.     

变形链球菌表面抗原Ⅰ／Ⅱ基因体外扩增片段的Southern杂交分析

目的：研究扩增产物的特异性。方法：将变链菌ＮＧ８ｓｐａＰ基因１０１７ｂｐ扩增产物，采用随机引物标记法，使用α－３２ＰｄＣＴＰ标记成ＤＮＡ探针，与１３株变链菌相应的扩增产物进行Ｓｏｕｔｈｅｒｎ杂交。结果：６株有强杂交信号，与ＰＣＲ结果一致，结论：说明扩增产物是特异的     

Development and clinical application of DNA probe specific for Peptostreptococcus micros

P. micros is thought to be an important pathogen in the etiology of certain inflammatory lesions, however, the role of this microorganism is uncertain due to the lack of rapid and reliable method to identify this species. The purpose of this study was to develop a DNA probe specific for the detection of P. micros in order to evaluate its prevalence in oral infectious lesions. The whole genomic DNA from P. micros was partially digested and inserted into the vector pUC 13. Four recombinant clones were selected, purified and screened against reference strains of Peptostreptococcus species to check the species specificity and then applied to clinical isolates. The sensitivity and specificity of the DNA probe for P. micros was 99.2% and 100%, respectively. P. micros could be detected in 7.2% of subgingival dental plaques from the patients with adult periodontitis and in 16.1% of the endodontic lesions with periapical pathosis. In the endodontic lesions, there was a good correlation between the clinical symptoms and the presence of P. micros. These data strongly suggested that the DNA probe can be useful in the detection of P. micros and that this microorganism is important in certain periodontal and endodontic lesions.     

牙龈卟啉单胞菌在龈下菌斑中的分布

研究牙龈卟啉单胞菌在牙周炎患者病变部位和健康部位龈下菌班中的分布情况,方法：选择６４例成年牙周炎患者,取龈下菌斑,经厌氧培养,挑战产黑色素菌落,经多聚酶链反应鉴定牙龈卟啉单胞菌。结果：产黑色素Ｇ＾－厌氧杆菌和牙龈卟啉单胞菌的患者检出率分别是６７．２％和６０．９％。牙龈卟啉单胞菌在病变部位和健康部位的检出率分别是３５．９％和２８．１％,二者差异无统计学意义;牙龈卟啉单胞菌在病变部位和健康部位的检出株     

Quantitative real-time polymerase chain reaction based on single copy gene sequence for detection of periodontal pathogens

OBJECTIVE: To develop a method for quantification of Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf) from subgingival plaque samples based on TaqMan real-time polymerase chain reaction (PCR) technology. MATERIAL AND METHODS: Bacterial cells from these species were obtained after culturing reference strains and were counted microscopically. Cellular suspensions in Tris-EDTA buffer were used for DNA extraction after boiling for 20 min. Primers for PCR were selected from sequences of the LktC (Aa), Arg-gingipain (Pg) and BspA antigen (Tf) genes in order to yield amplicons below 100 bp. TaqMan-based real-time PCR was adjusted to quantify each species separately. Cycle threshold (C(T)) values were calculated for each species according to the initial number of copies. A reliability analysis was carried out using intra-class correlation coefficients (ICCs) with a two-way random effects model. RESULTS: A high sensitivity and specificity was obtained for the detection of the three bacterial species. The TaqMan real-time PCR technology yielded a good repeatability in the obtained cycle threshold (C(T)) values for each initial number of copies, demonstrating coefficients of variation below 5% for each bacteria. The reproducibility of the technique was also demonstrated by the high ICCs (>0.98; p<0.00001) obtained for each bacteria with and without the addition of subgingival plaque. CONCLUSION: A novel diagnostic method based on TaqMan real-time PCR was developed for the quantification of Aa, Pg and Tf. It has demonstrated good sensitivity and repeatability on pure cultures. Its diagnostic utility should be demonstrated in subgingival plaque samples.     

Use of randomly cloned DMA fragments for the identification of oral spirochetes

DNA probes were produced for the detection and identification of 4 cultivable species of oral spirochetes, Treponema denticola, Treponema socranskii, Treponema vincentii and Treponema pectinovorum. To obtain probe sequences, chromosomal DNA, isolated from representative strains within each species, was cloned in Escherichia coli K-12. Cloned DNA fragments were screened for the ability to hybridize to DNA only from homologous strains. Several such fragments were identified and shown to be specific when tested against a series of DNAs from gram-negative and gram-positive oral bacteria. The selected probe sequences were semi-conserved within strains of T. denticola and T. socranskii such that restriction fragment length polymorphism (RFLP) was observed. In the case of T. socranskii, RFLP was useful in distinguishing between the 3 known subspecies. Chromosomal DNA fragments from 2 strains of T. vincentii failed to cross-hybridize, under stringent conditions, to genomic DNA from each of these strains. The hybridization probes were suitable for the identification of clinical isolates of T. denticola and could be used to detect the presence of individual Treponema species in mixed cultures. On this basis, the probes were used successfully to detect T. denticola in uncultured plaque samples.     

非核素标记牙龈卟啉菌核酸探针及其对比研究

采用非核素地高辛标记法标记Ｐｇ探针并与３２Ｐ标记法进行对比研究。地高辛标记采用随机引物法。通过观察碱性磷酸酶对ＮＢＴ和ＢＣＩＰ的催化反应产生蓝紫色斑点来判定阳性信号。结果表明：地高辛标记探针的特异性和敏感性与３２Ｐ标记的水平相当，且前者还兼有性能稳定、利于存放、可重复使用、无需考虑个人防护和废物处理等优点。