变形链球菌表面抗原Ⅰ／Ⅱ基因片段的聚合酶链反应扩增

目的：扩增变形链球菌表面抗原Ⅰ／Ⅱ基因片段，并作特异性鉴定。方法：根据其结构基因ｓｐａＰ序列设计１对寡核苷酸引物，采用聚合酶链反应（ＰＣＲ）方法扩增了其中长１０１７ｂｐ的片段。结果：所设计的引物能从６株变形链球菌标准株和部分口腔常居菌中扩增出大小一致的ＤＮＡ片段，结论：该基因部分序列广泛存在于变形链球菌和部分口腔常居菌，为研究口腔细菌的微生态关系提供了新的信息     

多聚酶链反应检测牙龈卟啉单胞菌

近年来，随着Ｐｇ菌分子生物学研究工作的深入，已有Ｐｇ菌纤毛基因得以分离并测序。本文根据Ｐｇ菌特异的纤毛亚单位旦白结构基因设计寡苷酸引物，采用ＰＣＲ技术增了其中１３１ｂｐ的片段，并对其进行特异性鉴定。     

多聚酶链反应检测牙周病人及其家庭成员中的牙龈卟啉单胞菌

近年来随着分子生物学的发展，发现作为牙周主要致病菌的牙龈卟啉单胞菌与其它产黑菌之间无同源性、证实其有特异性。作者根据牙龈卟呆单胞菌特定的纤毛亚单位蛋白结构的基因，设计一个寡核苷酸引物，采用多聚酶链反应简称ＰＣＲ扩增了１３ｂＰ特异片段，实验表明，ＰＣＲ直接检测临床标本中中的牙龈卟啉单胞菌，不仅特异、敏感，而且快速，从而显著了较好的优越性。同时，作者也证实该引物具有高度的特异性，可用此对此物检测口腔中     

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。     

变形链球菌蔗糖酶Ⅱ基因体外扩增片段的Southern杂交,序？…

将变链菌ＮＧ８ＳｃｒＡ基因４３２ｂｐ扩增片段纯化后，采用随机引物法标记成ＤＮＡ探针，与１３株变链菌相应的扩增产物进行Ｓｏｕｔｈｅｒｎ杂交，结果均出现杂交信号。进一步采用不对称ＰＣＲ法测序，结果与文献报道的序列一致。表明ＰＣＲ扩增产物特异性好，ＳｃｒＡ基因在变链菌及部分口腔常居菌间存在广泛同源性。     

变形链球菌蔗糖酶Ⅱ基因片段的PCR扩增（一）

根据变形链球菌（以下简称变链菌）ＧＳ５ＳｃｒＡ基因核苷酸序列，设计并合成了一对寡核苷酸引物，采用ＰＣＲ方法对１３株变链菌、２３株口腔常居菌进行扩增，结果全部变链菌及部分口腔常居菌均出现４３２ｂｐＤＮＡ片段。说明该基因部分序列广泛存在于变链菌及部分口腔常居菌，这为研究口腔细菌的微生态关系提供了新的信息。     

随意引物PCR用于口腔链球菌基因分型的初步研究

目的：探讨口腔链球菌基因分型，比较细菌相互间基因差异。方法：设计２对１０碱基随意引物，提取变链球菌、血链球菌染色体ＤＮＡ，分别用一对引物进行ＰＣＲ扩增。结果：变链球菌、血链球菌扩增的ＤＮＡ基因片段有较大区别。设计不同序列引物，扩增的ＤＮＡ片段亦不同，可以分辨不同的基因位点。结论：随意引物ＰＣＲ检测方法稳定，可作为细菌基因分型较好的方法     

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。     

口腔疣状癌中人乳头瘤病毒感染

本研究采用聚合酶链反应技术（ＰＣＲ），利用人乳头瘤病毒（ＨＰＶ）公共引物及ＨＰＶ１６型特异引物分析了１２例口腔疣状癌中ＨＰＶ的感染．结果表明９／１２例公共引物ＰＣＲ呈ＨＰＶ相关序列阳性；４／１３例ＨＰＶ１６特异性引物ＰＣＲ结果阳性，说明ＨＰＶ参与了口腔疣状癌的发生     

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     

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。     

牙龈卟啉菌核酸探针的制备,鉴定

根据Pg的特异的fimA基因设计一对引物,采用PCR技术扩增其中542bp长的DNA片段,用~(32)P标记作为探针与25株细菌DNA进行杂交鉴定.结果显示该探针能识别5株同种菌,而与其它20株异种菌DNA无阳性信号出现,探针的敏感性达到微微克级.提示该探针具有特异、敏感、快速、简便的特性.适用于对Pg的检测.     

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。     

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.     

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.     

牙龈卟啉菌及其胶原酶基因遗传多样性的研究

目的 分析牙龈卟啉单胞菌细菌基因型及其重要毒力因子胶原酶基因PrtC的遗传异质性，了解细菌遗传变异与其致病性的相关性。方法 对从不同牙周炎患者口腔中分离出的牙龈卟啉菌进行DNA指纹分析，参考菌株为PgATCC33277。通过PCR反应，检测临床菌株中是否存在特异性的胶原酶基因片段（548bp）。并通过酶切鉴定和DNA序列分析，了解PrtC基因遗传多样性的变化。结果 80株牙龈卟啉菌共获得7种基因型（Ⅰ-Ⅶ）。所有24株临床菌株和参考菌株PgATCC3327中均扩增出特异性的胶原酶基因片段。对照菌株中没有得到相应的产物。4株细菌的序列分析结果与国外的报道相比较，发现一些基因序列存在差异，出现6个核苷酸碱基缺失。结论 临床分离的牙龄卟啉菌中可检测到多种基因型，且具有个体差异，这些菌株具有合成胶原酶的能力，不同菌株胶原酶基因之间存在遗传异质性的变化。     

Identification of oral species of the genus Veillonella by polymerase chain reaction

Introduction:  Members of the genus Veillonella cannot be reliably distinguished by their biochemical characteristics and phenotypic features. Moreover, DNA–DNA hybridization and sequence analyses of the 16S ribosomal RNA gene including random fragment length polymorphism analysis, are complex and time-consuming procedures that are not well-suited to identifying oral species of Veillonella : Veillonella atypica , Veillonella denticariosi , Veillonella dispar , Veillonella parvula , and Veillonella rogosae .
Methods:  In this study, five forward primers and a reverse primer were designed for polymerase chain reaction (PCR) according to the partial sequences of the rpoB genes of these oral Veillonella species.
Results:  The forward primers were species-specific for these five Veillonella species, and could produce specific amplicons when used together with reverse primer and individual DNA templates of these species in PCR. These primer pairs were also found to discriminate between the respective species, and the Veillonella strains isolated from human oral cavities were successfully assigned to one of the five oral species of the genus Veillonella based on their specific products by PCR.
Conclusion:  A simple two-step PCR procedure using the five sets of primer pairs developed in the present study is a rapid and reliable method for the identification of the recognized oral Veillonella species.     

Detection of Helicobacter pylori DNA in recurrent aphthous stomatitis tissue by PCR

Helicobacter pylori is recognised as being an aetiological agent of chronic active gastritis and peptic ulcer disease and has been associated with an increased risk of gastric cancer. The natural reservoir for H. pylori is unknown, although the oral cavity has been the focus of much attention in this respect. Given the histological similarities between gastric and oral ulceration, it seemed prudent to investigate a possible association between H. pylori and recurrent aphthous stomatitis (RAS). In this study, the potential involvement of H. pylori in the aetiology of RAS was investigated using the polymerase chain reaction (PCR). Biopsies from 28 RAS patients were analysed, in addition to 20 oral lichen planus (OLP) and 13 normal biopsies that were used as controls. Genomic DNA was extracted from biopsies, and confirmation of successful extraction of PCR-amplifiable DNA was achieved by carrying out PCR on each DNA sample with nested primers specific for the human beta-haemoglobin gene. PCR identification of H. pylori was carried out using a primer pair specific for the H. pylori 16S ribosomal RNA (rRNA) gene. Two rounds of PCR were carried out to amplify a 295-bp product, and the identity of amplified products was confirmed by DNA sequencing. H. pylori DNA was detected in 3 of 28 (11%) RAS samples but not in any of 20 OLP and 13 normal samples. These results do not support a definitive aetiological role for H. pylori in RAS, although the possibility that H. pylori may be involved in a small proportion of RAS cases cannot be excluded.     

采用PCR方法鉴别伴放线放线杆菌的6种血清型

目的：探索采用PCR的方法对伴放线放线杆菌的不同菌株进行血清型分类。方法：根据伴放线放线杆菌不同血清型特异性多糖抗原基因序列设计6对不同的寡核苷酸引物，用这6对引物分别对所选择伴放线放线杆菌6种不同的血清型菌株各3株，共18株，其中参考菌株6株，系ATCC29523（血清型a），ATCC43718（血清型b），ATCC33384（血清型c），IDH781（血清型d），IDH1705（血清型e）以及CU1000（血清型f），其余12个菌株均为临床分离株的DNA进行PCR扩增分析。结果：每一对引物均针对相应的血清型产生特异性单一条带PCR产物，产物大小分别为428bp（a），298bp（b），559bp（c），690bp（d），211bp（e），232bp（f）。全部18个菌株均能够被准确识别，无交叉反应。结论：PCR方法可以快速准确地鉴别伴放线放线杆菌目前已知的全部6种血清型。