应用PCR检测慢性牙周炎患者龈下菌斑中牙龈卜啉单胞菌及感染菌株基因型的分析

目的 建立慢性牙周炎（CP）龈下标本中牙龈卟啉单胞菌（Porphyromonas gingivalis,Pg)的PCR检测方法，了解不同患牙龈下菌斑中Pg基因型的差异。方法 采用培养法分离不同患牙龈下菌斑中Pg，同时采用PCR进行Pg 16S rDNA基因、prtC基因和fimA基因的检测。部分扩增产物T－A克隆后测定核苷酸序列。结果 61例患者122个龈下菌斑标本中，Pg 16S rDNA、prtc和fimA分别扩增的阳性率依次为90．6％、81．9％和78．0％，联合扩增的阳性率高达98．4％，培养法阳性率仅为31．1％。30．0％（18／60）患者不同牙位龈下菌斑中的Pg基因型不一致。Pg 16S rDNA、prtC和fimA扩增片段与文献报道的核苷酸序列比较，同源性98．62％－100％。结论 所建Pg PCR检测方法具有较高的敏感性和特异性，可用于慢性牙周炎龈下标本中Pg的快速临床诊断。部分CP患者可被不同基因型的Pg菌株同时感染。     

rpoB作为蜡样芽胞杆菌群快速检测的标志基因的实验研究

目的探讨常规PCR和实时荧光定量PCR（Q-PCR）方法检测蜡样芽胞杆菌群rpoB基因的特异性和敏感性。方法提取蜡样芽胞杆菌群和其他各种对照细菌的基因组DNA,合成蜡样芽胞杆菌群rpoB基因扩增引物,采用常规PCR和SYBRgreen实时定量PCR两种方法扩增rpoB基因片段,并将PCR产物克隆到pMD18-T载体后进行DNA测序。结果常规PCR和Q-PCR均能扩增出蜡样芽胞杆菌群rpoB基因的174bpDNA片段,而各种对照菌株均未见扩增。序列比对发现蜡样芽胞杆菌群细菌在该片段中存在5处核苷酸的不同,差异率为2.88%。以炭疽芽胞杆菌基因组DNA系列稀释作为扩增模板显示常规PCR最小检出量为3.42pg,Q-PCR的敏感性达到171fg,3次重复实验显示Q-PCR检测rpoB基因的灵敏度为（3.32×101±7.45×100）拷贝。结论以rpoB基因为检测靶基因的Q-PCR方法具有高度的特异性和良好的敏感性,能实现对蜡样芽胞杆菌群快速而准确的检测。     

牙龈卟啉单胞菌prtH基因分布与多态性研究

目的　分析牙龈卟啉单胞菌 (P .gingivalis)prtH基因的遗传多态性 ,从而了解细菌变异与其对牙周致病作用的相关性。方法　以PCR技术从牙周炎患者口腔中分离的P .gingivalis进行prtH基因扩增 ;采用斑点杂交法 ,以生物素标记prtH基因为探针 ,与以引物B进行PCR扩增为阳性的P .gingivalis基因组DNA杂交 ;对PCR产物进行AluⅠ限制性酶切和DNA测序分析。结果　以prtH基因A、B引物对 14 0株临床分离株进行PCR扩增 ,分别在 76例 (引物A)和 117例 (引物B)出现阳性扩增产物 ,阳性率分别为 5 4 .2 %、84 %。对 34例引物B扩增阳性的P .gingivalisDNA进行点杂交检测 ,阳性吻合率为 82 .4 %。与GenBank中菌株ATCC332 77(U15 2 82 )和W83(L2 74 83)的prtH基因序列相比 ,5株P .gingivalisPCR扩增产物的测序存在着缺失和点突变。结论　在慢性牙周炎患者临床P .gingivalis分离菌株中存在着prtH基因的遗传多态性 ,本实验建立了具有高度敏感性和特异性的PCR检测方法     

人工耳蜗植入者线粒体12SrRNA基因突变的分析

目的研究接受人工耳蜗植入的耳聋患者中，线粒体12SrRNA基因突变的类型和发生概率。方法选取100例接受人工耳蜗植入的非综合征性耳聋患者（语前聋96例，语后聋4例；氨基糖苷类药物使用史者16例），取外周血提取基因组DNA，以PCR方法扩增线粒体12SrRNA基因，扩增产物纯化后直接测序分析突变。结果100例患者中有2例检测到线粒体12SrRNA基因1555A〉G纯合突变，1例检测到delT961Cn杂合突变，致病基因突变总检出率为3％。结论在所研究的人工耳蜗植入群体中，线粒体12SrRNA基因突变不是主要的致聋病因。     

变性高效液相色谱技术快速检测下呼吸道致病性细菌的实验研究

目的 运用变性高效液相色谱(DHPLC)技术检测5种下呼吸道致病性细菌,建立一种快速检测下呼吸道致病性细菌的分子生物学方法.方法 以下呼吸道致病性细菌16S rRNA基因的保守区设计通用引物,并在引物前加上40-bpGC,特异性扩增该基因的保守区和可变区,运用DH-PLC技术对PCR产物进行分析.选取50株临床分离菌株验证该方法的有效性.结果 通用引物可特异性扩增细菌16S rRNA,PCR产物经DHPLC分析后每种细菌均能得到特征性的洗脱峰.DHPLC检测临床分离菌株结果显示,与常规培养方法的符合率为100%.结论 DHPLC技术具有准确、简便、快捷和高通量等特点,在临床检测下呼吸道致病性细菌中具有潜在的应用价值.     

慢性牙周炎患者龈下菌斑中伴放线放线杆菌基因型的分析

目的：建立龈下菌斑标本中伴放线放线杆菌（Actinobacillus actinomycetemcomitans，Aa）PCR检测方法，了解慢性牙周炎患者不同牙位的龈下菌斑中Aα 的感染率及其优势基因型。方法：61例慢性牙周炎患者每例采取2个不同牙位共122份龈下菌斑标本，采用培养法分离Aα菌株，以PCR或多重PCR检测16SrDNA基因、lktA基因和fap基因，部分扩增产物克隆后测序。结果：在11例患者的11份龈下菌斑标本中分离到Aα菌株。122份龈下菌斑中Aα16SrDNA、lktA、和fap检测阳性率分别为84.4%、75.4%、和50.0%。38.8%的患者（19/49）不同牙位龈下菌斑中检出的Aα基因型不一致。Aα有4种基因型，其优势基因型是16SrDNA^ /lktA^ /fap^ ，其次为16SrDNA^ /lktA^ /fap^-。部分标本上述3种基因的扩增片段与文献报道核苷酸序列的同源性为93.75%-100%。结论：建立的PCR或多重PCR有较高的敏感性和特异性，适用于龈下菌斑标本中Aα的快速检测。慢性牙周炎患者Aα感染率较高，并存在优势基因型，部分患者可被不同基因型的菌株同时感染。     

长臂光敏生物素和光敏地高辛核酸探针检测医学真菌灵敏度的比较研究

建立应用长臂光敏生物素核酸探针和光敏地高辛核酸探针杂交。检测真菌的方法，并比较二者检测临床常见医学真菌的敏感性。设计并合成医学真菌通用引物，用PCR扩增白色念珠菌标准株的核糖体RNA基因，用长臂光敏生物素或光敏地高辛标记其纯化的扩增产物制备成相应的核酸探针，然后用上述两种探针对标本中的PCR扩增产物进行Southern杂交，检测医学真菌。通过用白色念珠菌感染大鼠，建立念珠菌病的实验动物模型，验证血培养法、PCR法和PCR扩增结合Southern杂交，检测真菌的敏感性。结果表明，这两种核酸探针可分别与白色念珠菌、热带念珠菌、新型隐球菌、黄曲霉菌、烟曲霉菌等9种真菌杂交呈阳性结果：与临床常见的细菌、病毒和哺乳动物组织细胞DNA杂交结果为阴性。利用实验动物模型比较了血培养法、PCR法和PCR扩增结合长臂光敏生物素核酸探针Southern杂交法检测真菌血症的灵敏度，实验证明后者的敏感性最高。总之，应用长臂光敏生物素核酸探针和光敏地高辛核酸探针与标本PCR扩增物杂交，检测上述真菌既特异和敏感，又不需放射性同位素。光敏地高辛核酸探针检测医学真菌的灵敏度略高于长臂光敏生物素核酸探针。     

聚合酶链式反应非特异性扩增抑制因子的发现以及全血直接PCR检测G6PD基因外显子3—4突变

目的（1）研究消除PCR反应非特异性扩增新技术。（2）研究全血直接PCR能否用于葡萄糖6-磷酸脱氢酶基因突变检测。方法（1）将新鲜全血混合基因组DNA进行PCR扩增,看全血能否抑制基因组DNA非特异性扩增。将新鲜人血清混合基因组DNA进行PCR扩增,看新鲜人血清能否抑制基因组DNA非特异性扩增。（2）将1μl全血加入PCR反应液,98℃变性后转入PCR循环,使用Taq（Fermentas）扩增G6PD基因外显子3—4,取扩增产物50μl进行DNA双向自动测序,使用ClustalX软件将测序结果与基因库中标准G6PD基因外显子3—4序列进行比对分析。结果（1）发现新鲜全血能抑制基因组DNA非特异性扩增,进一步研究发现是新鲜人血清抑制了基因组DNA非特异性扩增。（2）全血直接PCR加上DNA测序找到一例G6PD突变g．13503A〉G。结论（1）新鲜人血清中存在PCR非特异性扩增抑制因子。（2）使用TaqDNA聚合酶（Fermentas）能进行全血直接PCR扩增,加上DNA测序可用于G6PD基因突变研究。     

细胞培养中细菌类微生物污染的快速检测

目的 检测细胞培养中细菌类(包括支原体)微生物的污染情况。方法 用细胞培养中常见的培养物人为污染He-La细胞，设计细菌类微生物16S rRNA基因序列通用引物，PCR扩增16SrRNA基因序列片断，建立PCR检测培养细胞污染的方法，并用该方法检测本室保存的细胞株的污染情况。结果 人为污染绿脓杆菌、大肠杆菌、白色葡萄球菌和支原体M.fernentans的Hela细胞的培养上清中均扩增出大小的片段，与目的片段相符。本室所收藏的15个细胞株有3株的培养上清中扩增出大小的片段。结论 本实验建立了：16SrRNA基因序列通用引物PCR法，可用于快速检测培养细胞中细菌类微生物的污染。     

膜芯片技术检测泌尿生殖道沙眼衣原体、淋病奈瑟菌和3种支原体的方法学研究

目的建立和评价一个新的多重PCR．反向线点杂交技术（RIJB）快速同时检测泌尿生殖道沙眼衣原体（Ct）、淋病奈瑟菌（坛）和3种支原体感染的方法。方法分别选择Ct隐蔽质粒和Ng16SrRNA基因设计两对特异性引物，以支原体内转录间隔序列（ITS）设计一对支原体属通用引物，生物素标记下游引物。构建三重PCR同时扩增Ct、Ng、解脲脲原体（仇）、微小脲原体（跏）、人型支原体（Mh）等菌DNA，然后与固定在尼龙膜上的各特异性寡核苷酸探针杂交。并对142份经荧光定量PCR（FQ-PCR）检测0和坛的性病高危人群标本，以及45份经支原体液体培养法鉴定的标本进行检测。结果多重PCR可同时扩增Ct、Ng、Uu、Up和Mh标准菌株DNA，其PCR产物的片段长度为208～408bp。97份FQ-PCRCt阳性标本中有93份经多重PCR-RLB检测为Ct阳性，45份FQ-PCRCt阴性标本中35份多重PCR-RLB阴性。41份FQ-PCRNg阳性标本中34份多重PCR-RLBNg阳性，101份FQ-PCRNg阴性标本中98份多重PCR-RLB阴性。其中36份经多重PCR-RLB检测为混合感染。32份支原体液体培养阳性标本中28份多重PCR-RLB阳性，13份支原体培养阴性标本经多重PCR-RLB检测均为阴性。结论多重PCR-RLB可快速同时检测Ct、Ng、Uu、Up和Mh，为性传播疾病的临床诊断提供了一种可靠的方法。     

Nested PCR Assay for Detection of Granulocytic Ehrlichiae

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.     

应用单一和双重荧光定量PCR法快速检测军团菌

目的 建立单一和双重荧光定量PCR方法分别和同时进行军团菌属及嗜肺军团菌的检测.方法 利用军团菌属16 S rRNA基因和嗜肺军团菌mip基因设计引物和探针,两条基因探针分别标记FAM和HEX,并将相关反应体系和条件进行优化.分别应用单一基因探针(单一荧光定量PCR)和双重基因探针(双重荧光定量PCR)对嗜肺军团菌、非嗜肺军团菌及非军团菌进行检测,并验证两种方法的特异度、敏感度.应用双重荧光定量PCR检测空调水样滤膜样品和DNA提取样品,比较两者结果的一致性.结果 针对军团菌属及嗜肺军团菌,应用荧光定量PCR,16 S rRNA基因和mip基因均能较好的检出,16S rRNA和mip的最低检出限分别为8和10个拷贝.经优化得到了最佳反应体系.单一荧光定量PCR方法所检的8株嗜肺军用菌及4株非嗜肺军团菌16 S rRNA基因均为阳性,嗜肺军团菌mip基因阳性,非嗜肺军团菌mip基因阴性.双重荧光定量PCR方法所检的23株嗜肺军团菌中有2株为假阴性,9株非嗜肺军团菌和非军团菌属中有1株为假阳性.49份空调水样滤膜直接检测和提取DNA后检测的结果一致,其中26份水样军团菌阳性,20份为嗜肺军团菌,6份为非嗜肺军团菌;1份弗朗西斯菌检测HEX阳性(假阳性),占实际培养分离的1/26.结论 单一及双重荧光定量PCR法特异、快速、敏感,一次同时检测嗜肺与非嗜肺军团菌,满足对空调和环境水样军团菌监测的要求.     

应用单一和双重荧光定量PCR法快速检测军团菌

目的 建立单一和双重荧光定量PCR方法分别和同时进行军团菌属及嗜肺军团菌的检测.方法 利用军团菌属16 S rRNA基因和嗜肺军团菌mip基因设计引物和探针,两条基因探针分别标记FAM和HEX,并将相关反应体系和条件进行优化.分别应用单一基因探针(单一荧光定量PCR)和双重基因探针(双重荧光定量PCR)对嗜肺军团菌、非嗜肺军团菌及非军团菌进行检测,并验证两种方法的特异度、敏感度.应用双重荧光定量PCR检测空调水样滤膜样品和DNA提取样品,比较两者结果的一致性.结果 针对军团菌属及嗜肺军团菌,应用荧光定量PCR,16 S rRNA基因和mip基因均能较好的检出,16S rRNA和mip的最低检出限分别为8和10个拷贝.经优化得到了最佳反应体系.单一荧光定量PCR方法所检的8株嗜肺军用菌及4株非嗜肺军团菌16 S rRNA基因均为阳性,嗜肺军团菌mip基因阳性,非嗜肺军团菌mip基因阴性.双重荧光定量PCR方法所检的23株嗜肺军团菌中有2株为假阴性,9株非嗜肺军团菌和非军团菌属中有1株为假阳性.49份空调水样滤膜直接检测和提取DNA后检测的结果一致,其中26份水样军团菌阳性,20份为嗜肺军团菌,6份为非嗜肺军团菌;1份弗朗西斯菌检测HEX阳性(假阳性),占实际培养分离的1/26.结论 单一及双重荧光定量PCR法特异、快速、敏感,一次同时检测嗜肺与非嗜肺军团菌,满足对空调和环境水样军团菌监测的要求.     

Evaluation of culture methods and a DNA probe-based PCR assay for detection of Campylobacter species in clinical specimens of feces

Campylobacter species are the leading agents of bacterial gastroenteritis in developed countries. In this study 320 specimens of feces from patients with symptoms of acute gastroenteritis were cultured for Campylobacter species by direct plating on modified charcoal cefoperazone deoxycholate agar and by enrichment in modified Preston broth, with or without blood added, for 48 h at 37 degrees C prior to plating. A 16S/23S PCR/DNA probe membrane-based colorimetric detection assay was evaluated on a subset of the feces (n = 127), including 18 culture-positive and 109 culture-negative specimens. DNA was extracted directly from the fecal specimens by using the QIAamp DNA stool Minikit for the DNA probe-based PCR assay (PCR/DNA probe assay). A second PCR/DNA probe assay based on the 16S rRNA gene in Campylobacter spp. was applied to all specimens that were culture negative, PCR/DNA positive on initial analysis. Campylobacter species were cultured in 20 of the 320 specimens. The 16S/23S PCR/DNA probe assay detected campylobacter DNA in 17 of 18 (94% sensitivity) culture-positive specimens and in 41 (38%) culture-negative specimens. The presence of campylobacter DNA in 35 of 41 culture-negative specimens was confirmed by the 16S PCR/DNA probe assay. DNA sequence analysis of seven 16S/23S PCR products and five 16S PCR products amplified from a selection of these specimens confirmed the presence of campylobacter DNA and more specifically Campylobacter jejuni, C. concisus, C. curvus, and C. gracilis DNA in these specimens. The molecular assays described in this study are rapid methods for the detection and identification of Campylobacter species in fecal specimens. The finding of Campylobacter spp. DNA in a large number of specimens of feces from patients with no other identified cause of diarrhea may suggest that Campylobacter spp. other than C. jejuni and C. coli may account for a proportion of cases of acute gastroenteritis in which no etiological agent is currently identified.     

口腔黏膜拭子在遗传性耳聋常见致病基因检测中的应用研究

目的：探讨口腔黏膜拭子在遗传性耳聋常见致病基因检测中的应用,为常见耳聋致病基因筛查提供无创的样本采集方式。方法：195例新生儿,经听力筛查被诊断为听力障碍,收集其口腔黏膜拭子和外周血样本,抽提DNA,核酸测定仪检测两种收集方式样本所得DNA质量。对两种样本进行三种常见遗传性耳聋致病基因GJB2基因、SLC26A4基因和线粒体基因12S rRNA全外显子巢式PCR扩增,比较两种方法扩增产物及测序效果。结果：口腔黏膜拭子抽提DNA质量较外周血低,差异具有统计学意义（P〈0．05）;在PCR扩增后,两种样本扩增产物凝胶电泳无明显差异,测序结果无明显差异（P〉0．05）。结论：口腔黏膜拭子作为一种无创的样本采集方式,可用于三种常见遗传性耳聋致病基因检测的样本采集。     

Detection of bacterial DNA in cerebrospinal fluid by an assay for simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and streptococci using a seminested PCR strategy.

Primers specific to conserved and variable regions in the 16S rRNA sequence were selected from the partially sequenced 16S rRNA genes of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, S. agalactiae, and Staphylococcus epidermidis. The PCR assay was divided into two DNA amplifications. The first resulted in a general bacterial amplicon, and the second resulted in a species-specific amplicon. The high specificity of the PCR assay was documented after testing bacteria of 28 different species (133 strains). A total of 304 clinical cerebrospinal fluid samples, including 125 samples from patients with bacterial meningitis, were assayed to investigate the diagnostic sensitivity and specificity for bacterial meningitis. The assay showed high sensitivity (0.94) and specificity (0.96) with the clinical samples, although some false results were obtained, the reasons for which are discussed. With agarose gel electrophoresis for detection of the PCR products, the detection limit for meningococci in cerebrospinal fluid was 3 x 10(2) CFU/ml.     

Comparison of a new quantitative ompA-based real-Time PCR TaqMan assay for detection of Chlamydia pneumoniae DNA in respiratory specimens with four conventional PCR assays

Chlamydia pneumoniae, an important respiratory pathogen, is difficult to culture, and detection rates by conventional PCRs vary considerably. A new quantitative ompA-based real-time PCR assay based on TaqMan technology for detection of C. pneumoniae in respiratory samples is described, and its performance in terms of sensitivity and reproducibility is compared with those of four published conventional PCRs (one single-step PCR targeting a cloned PstI fragment; two nested PCRs, one targeting the 16S rRNA gene followed by hybridization and the other targeting the ompA gene; and a touchdown enzyme time-release [TETR] PCR also targeting the 16S rRNA gene). Both ompA-based PCRs showed the best analytical sensitivity. All five assays could detect even lower target levels from spiked sputum, with the 16S rRNA assays performing better than the ompA-based nested PCR (10(-6) inclusion-forming units [IFU] were detected in four of four and two of four replicates by the 16S rRNA TETR PCR and the 16S rRNA nested PCR, respectively). In general, the ompA-based real-time protocol produced the most consistent positive results for all replicates tested down to 10(-6) IFU. Eight of 45 patient sputum specimens (18%) were C. pneumoniae DNA positive in at least one of four replicates tested by at least one assay. Without taking into consideration the analytical sensitivity or the reproducibility of the test results, the numbers of C. pneumoniae DNA-positive sputum specimens (n = 8) were four, three, two, two, and one for the 16S rRNA TETR assay, the PstI-based single-step PCR, the ompA-based real-time PCR, the ompA-based nested touchdown PCR, and the 16S rRNA-based nested PCR, respectively. However, the overall rate of concordance of positive results was low. Only one cell culture-positive sputum specimen was positive by four of five assays (14 of 16 replicates; mean cycle threshold value, 25; 10(8) particles/ml of sputum). Thirty-seven specimens were C. pneumoniae negative by all five assays for all replicates tested, as were all negative controls (n = 65 to 100 per testing panel). No PCR inhibitors were detected by real-time PCR or by the 16S rRNA-based nested assay. We confirm that the analytical sensitivity of an assay for the detection of C. pneumoniae does not necessarily predict its ability to detect its target in sputum. A quantitative, fast, and easy-to-handle diagnostic approach such as the ompA-based real-time TaqMan PCR described here might improve the detection of C. pneumoniae in respiratory samples.     

Detection of Sporothrix schenckii in clinical samples by a nested PCR assay

Cutaneous sporotrichosis is a chronic granulomatous fungal infection caused by Sporothrix schenckii with worldwide distribution. Its traditional diagnosis is time-consuming and difficult to differentiate from that of a clinical sporotrichoid lesion caused by various pathogens. In this study, a nested PCR assay for the detection of S. schenckii was evaluated by using a sequence of 18S rRNA gene as a target. For the examination of specificity and sensitivity, five clinical isolates with 1 ATCC 10213 strain of S. schenckii, 10 strains of clinical common fungi, 3 strains of Mycobacterium spp., Staphylococcus aureus, and normal human skin tissue were used. The expected fragment was amplified from six S. schenckii isolates in the first round and nested PCR but not from other microorganisms and human DNA. Their sequences were 100% identical to the S. schenckii 18S rRNA gene sequence deposited in GenBank. A detection limit of 40 fg of S. schenckii DNA extract was determined with ethidium bromide staining. Serial dilution studies demonstrated that the nested PCR could detect a DNA amount of 1 CFU of S. schenckii in tissue samples. We further investigated the nested PCR assay for the detection of S. schenckii from the tail tissues of 5 experimentally infected mice and from the clinical biopsy specimens of 12 patients with sporotrichosis confirmed by culture or histochemical staining. The nested PCR assay was positive in all 5 infected mice and in 11 of the 12 clinical specimens. The high sensitivity and specificity of this nested PCR indicate that the assay can provide rapid diagnosis with sufficient accuracy to be clinically useful for patients with sporotrichosis.     

Comparison of four different primer sets for detection of Helicobacter pylori in gastric biopsies and oral samples by using real-time PCR

AIMS: The presence of Helicobacter pylori. in the oral cavity remains controversial and the most appropriate method for detection of oral H. pylori has yet to be established. The aim of the present study was to compare four different primer sets on the detection of H. pylori in gastric biopsies and oral samples using real-time PCR. MATERIAL AND METHODS: Gastric biopsy and oral samples were collected from eight patients with gastric symptoms. DNA from clinical samples was extracted and analyzed for the presence of H. pylori by real-time PCR (LightCycler 2.0) using four pairs of primers which targeted 16S rRNA (16S rRNA#295; 16S rRNA#120) or glmM (glmM#294; glmM#722) DNA genes. Three H. pylori strains and three clinical isolates served as reference. The specificities of the four primer pairs were examined for seven oral microorganisms and two Helicobacter non-pylori species. RESULTS: Primer pair 16S rRNA#120 showed an acceptable specificity and a high sensitivity. Primer pairs glmM#294 and glmM#722 demonstrated a high specificity but a low sensitivity and primer pair 16S rRNA#295 demonstrated a poor specificity but acceptable sensitivity. Four H. pylori positive gastric biopsies were demonstrated by culture, histology and real-time PCR with primer pairs 16S rRNA#295 or 16S rRNA#120. No H. pylori was detected in oral samples, either by culture or by real-time PCR. CONCLUSION: Of the four different primer pairs examined, 16S rRNA#120 was the most appropriate to detect H. pylori in clinical samples using real-time PCR.