幽门螺杆菌基因的检测与分型

我们用幽门螺杆菌(Ｈｐ)空泡毒素(ｖａｃＡ)和细胞毒相关蛋白(ｃａｇＡ)基因检测以及ｖａｃＡ基因分型的聚合酶链反应(ＰＣＲ)方法,并对幽门螺杆菌ｖａｃＡ基因型和ｃａｇＡ状态的关系作了调查,报告如下。一、材料和方法1标本:48株Ｈｐ临床分离株和124份用我院自制采样器所取Ｈｐ阳性胃粘液标本[1],经ＰＣＲ检测Ｈｐ16ＳｒＲＮＡ基因和尿素酶试验以及14Ｃ呼气试验确定为Ｈｐ阳性的标本。以上取样的172位Ｈｐ阳性患者中,经内窥镜取样病检诊断为萎缩性胃炎(ＡＧ ＣＡＧ)的患者为49例,浅表性胃炎(ＳＧ ＣＳＧ)的患者为64例。2.试剂:ＴａｑＤＮ…     

多重PCR在幽门螺杆菌检测及分型中的应用

应用多重ＰＣＲ同时扩增１６ＳｒＲＮＡ和ｃａｇＡ基因来检测幽门螺杆菌感染及其分型。ＰＣＲ产物经ＤＮＡ序列测定证实为转异性扩增，敏感度为１０＾２ＣＦＵ／ｍｌ。４８株Ｈｐ菌株中，Ｉ型菌株（ｃａｇＡ＋）２６株（５４．２％）；５５０份胃粘液标本，Ｈｐ阳性（１６ＳｒＲＮＡ基因＋）２１６份（４７．５％），其中Ｉ型Ｈｐ１３９份（５３．３％）。     

幽门螺杆菌与胆囊胆固醇结石形成关系的研究

目的 研究胆囊幽门螺杆菌感染与胆囊胆固醇结石形成的关系.方法 我院2002年6月至2004年1月肝胆外科腹腔镜胆囊切除术60例,采用对照研究方法,对35例单纯胆囊结石患者(实验组)及25例单纯胆囊息肉患者(对照组)的胃幽门螺杆菌进行检测,采用PCR方法检测2组患者的胆汁及结石幽门螺杆菌细胞毒素相关基因抗原(CagA).结果 实验组胃幽门螺杆菌感染率为51.43%(18/35),对照组为48.00%(12/25),2组患者胃幽门螺杆菌感染率差异无统计学意义(χ~2=0.079,P>0.05).实验组胆汁标本中CagA 7例阳性(20.00%),结石标本中1例阳性(2.86%),对照组胆汁标本无一例阳性,2组胆汁CagA检出率差异有统计学意义(χ~2=5.822,P<0.05).结论 幽门螺杆菌DNA存在于胆囊胆固醇结石患者的胆汁及结石中,与胃幽门螺杆菌感染有关,胆囊幽门螺杆菌感染与胆囊胆固醇结石形成有关.     

HP在胃镜常规清洗消毒方法中清除效果的临床观察

目的：了解目前常规清洗消毒胃镜方法－三步法对幽门螺杆菌（ＨＰ）清除效果。方法：采用聚合酶链反应（ＰＣＲ）及ＨＰ培养法对４２例患者胃镜检查后,清洗消毒前后的胃镜活检测通道冲洗液进行了检测。结果：３１例（７３．８％）为ＨＰ感染者,ＰＣＲ检测清洗消毒前冲洗液有２６份（８３．９％）为阳性,清洗消毒后仍有９份（３４．６％）为阳性;而培养在清洗消毒前有１２份（３８．７％）为阳性,清洗消毒后均为阴性。另有２例患     

聚合酶链反应结合探针杂交检测幽门螺杆菌

目的建立特异、灵敏的聚合酶链反应（ＰＣＲ）结合长臂光敏生物素探针杂交检测幽门螺杆菌（ＨＰ）的方法。方法用ＰＣＲ产物直接克隆并测序，以含ＨＰＤＮＡ序列的重组质粒外源片段为探针。用细菌培养、单纯ＰＣＲ和ＰＣＲ结合杂交（ＰＣＲ－Ｓｂ）检测经胃镜诊断为慢性胃炎、胃溃疡、十二指肠球部炎症、十二指肠溃疡的胃活检组织、唾液和粪便标本中的ＨＰ。结果检测胃活检标本７９份，ＰＣＲ－Ｓｂ阳性率为９９％，单纯ＰＣＲ及细菌培养的阳性率分别为９５％和６５％，ＰＣＲ－Ｓｂ的灵敏度达到１ｆｇ。唾液标本的检测阳性率为３５％，粪便标本的检测结果均为阴性。结论测序证实了ＰＣＲ扩增的正确性。ＰＣＲ结合长臂光敏生物素探针杂交是特异、灵敏、简便和对人体无害的检测ＨＰ的方法。     

聚合酶链反应检测粪便中的副溶血性弧菌的TDH基因

副溶血性弧菌是细菌性腹泻的主要致病因子，本研究建立一种快速标本处理和聚合酶链反应诊断方法，扩增副溶血性弧菌的热稳定直接溶血素基因。对一组５６例腹泻患者粪便便标本进行了检测，经细菌培养培养证实为副溶血性弧菌的９份标本，ＰＣＲ法均阳性；培养法阴性的３９例中，有７例检测到ＴＤＨ基因。统计学表明两种方法检测结果相当一致，但检出能力ＰＣＲ法显著高于培养法。本法简便，快速，特异，适宜临床应用。     

聚合酶链反应同时检测五种腹泻致病菌的初步研究

目的探索一种快速、经济、敏感的诊断方法，对常见的腹泻致病菌进行基因检测研究。方法建立一种快速标本处理和多重聚合酶链反应（ｍｕｌｔｉｐｌｅｘＰＣＲ）诊断方法，在一次ＰＣＲ反应中同时扩增５种菌的致病基因：志贺菌及侵袭性大肠埃希菌的侵袭性质粒（ｉａｌ）基因、副溶血性弧菌的热稳定直接溶血素（ｔｄｈ）基因、Ｏ１群霍乱弧菌的霍乱毒素Ａ亚单位（ｃｔｘＡ）基因和产不耐热肠毒素大肠埃希菌的不耐热肠毒素Ｂ亚单位（ＬＴ－Ｂ）基因。扩增产物经电泳，出现与已知阳性对照细菌条带大小一致的条带即判断为该标本阳性。结果对一组常规培养生化鉴定法诊断为霍乱的２６份腹泻患者标本检测后，ＰＣＲ法有２４份检测到ｃｔｘＡ基因，ＰＣＲ阴性的２份为非Ｏ１群；另一组５６份粪标本分别检测到了ｉａｌ、ｔｄｈ、ＬＴ－Ｂ基因，ＰＣＲ法较培养法阳性率高。结论该方法具有简便、快速、特异、经济和不需培养等特点，适于临床应用。     

口腔与胃内幽门螺杆菌感染的相关性研究

目的探讨口腔与胃幽门螺杆菌感染的相关性。方法用幽门螺杆菌的特异性引物及特异Taqman探针进行荧光定量聚合酶链反应(FQ-PCR)检测27例根除治疗失败患者牙菌斑和胃黏膜幽门螺杆菌DNA,并进一步运用单链构象多态性(SSCP)分析技术分析口腔和胃内幽门螺杆菌菌株DNA基因型。结果 FQ-PCR:27例胃黏膜及牙菌斑标本采用FQ-PCR检测幽门螺杆菌,胃黏膜均为阳性;牙菌斑25例阳性,2例阴性。SSCP:胃黏膜和牙菌斑幽门螺杆菌基因型相同者(SSCP带型相同)19例,其余6例两者至少有1种相同的SSCP带型。结论同一个体牙菌斑和胃黏膜中的幽门螺杆菌属同种菌株的可能性大。口腔幽门螺杆菌可能是胃幽门螺杆菌感染的重要来源。胃幽门螺杆菌感染与口腔幽门螺杆菌感染密切相关。     

PCR和巢式PCR直接检测血清中HIV核酸序列

采用ＰＣＲ和巢式ＰＣＲ方法对５３例ＨＩＶ抗体阳性和阴性标本进行了检测，以扩增ＨＩＶ特异性ＤＮＡ序列，结果美国ＡｂｂｏｔｔＨＩＶ阳性血清２份，新加坡阳性血清２份，国内阳性血清２份及ＨＩＶ－１病毒培养物１份，两套ＰＣＲ扩增反应物均为阳性，对其中一套ＰＣＲ扩增的ｇａｇ基因序列进行巢式ＰＣＲ也为阳性。采自西南边境地区的２３份ＥＬＩＳＡ和ＷＢ阳性血清，各种ＰＣＲ反应均为阳性；１１份ＥＬＩＳＡ可疑阳性、ＷＢ阴性标本中，有两例血清呈ＰＣＲ阳性，探针杂交亦为阳性。而１２例ＳＩＶ、ＳＲＶ及猴血清标本ＰＣＲ反应均为阴性。上述结果表明，采用ＰＣＲ方法检测血清中ＨＩＶ核酸序列是一种很有前途的ＨＩＶ感染诊断方法。     

PCR方法诊断重症急性胰腺炎继发全身性感染13例研究

目的 探讨以１６ｓｒＲＮＡ和１８ｓｒＲＮＡ基因为基础的细菌和真菌ＰＣＲ方法诊断重症急性胰腺炎继发全身性感染的价值。方法 对急性胰腺炎的血标本进行细菌、真菌通用引物ＰＣＲ检测,并同常规培养比较。结果 对于所收集１３例重症急性胰腺炎患的２２份血标本,细菌培养阳性１例,真菌培养阳性１例,阳性检出率分别为４．５％和４．５％;而细菌ＰＣＲ检测阳性有７例,真菌ＰＣＲ阳性为１份,阳性检出率分别为３１．８％和４     

PCR扩增16S rRNA基因检测幽门螺杆菌的特异性研究

为了评估 PCR扩增 1 6S r RNA基因检测幽门螺杆菌的特异性 ,对 Hp和非 Hp临床分离菌株、胃活检组织、胃黏液、大便、淋巴细胞及其它非胃组织进行了 Hp的检测。 49株 Hp均出现 1 0 9bp特异扩增条带 ,而 7株非 Hp菌株为阴性 ;77份胃活检组织中 ,细菌培养阳性 48份 ,PCR阳性 50份 ;550份吞线取样的胃黏液标本 PCR扩增 Hp阳性 2 61份 (47.5% ) ;2 0份淋巴细胞、2 0份大便、5份乳房组织、4份肌肉组织、7份肺组织、2份脾组织和 3份肝组织标本 ,PCR扩增均阴性。结果显示 ,PCR法扩增 1 6S r RNA基因有较高的特异性 ,可用于临床标本 Hp的检测。     

PC R 检测技术在幽门螺杆菌诊断检测中的应用探讨

目的探寻聚合酶联反应(PCR)检测技术在幽门螺杆菌(H .pylori ,Hp)检测中的应用.方法通过对该院消化内科疑是患胃或十二指肠溃疡、胃或胃窦炎、胃癌患者于胃镜下取其病变部位组织标本,进行 Hp分离培养、PCR体外扩增,患者同时做尿素酶试验、血清学抗体检查,将结果进行比较分析.结果170份病例标本中,分离培养67例、尿素酶试验82例、Hp抗体检查152例、PCR扩增144例阳性.阳性率分别为39.4%、48.2%、89.5%、84.7%.以细菌培养为金标准,则尿素酶试验、抗体检查、PCR灵敏度分别为88.1%、89.5%、98.5%,特异性分别为94.6%、77.4%、81.7%.这4种检测方法中,灵敏度最高的为PCR基因扩增法,其次为抗体检查和尿素酶试验;特异性最高的为尿素酶试验,其次为PCR扩增和抗体检查.结论 PCR试验特异性和灵敏度均较高,可以用作Hp感染的确诊、基因分型、流行病学调查、微量取材等的检查.     

Real-time quantitative PCR for the detection of Streptococcus pneumoniae in the middle ear fluid of children with acute otitis media

PCR based on the amplification of pneumolysin gene fragments has previously been applied to demonstrate Streptococcus pneumoniae in clinical specimens. Here, a real-time PCR method for the detection and quantification of pneumococci by amplifying a 206-bp fragment of the pneumolysin-encoding gene is described. The amplified fragments were detected simultaneously using fluorescent-labeled sequence-specific hybridization probes. The applicability of the assay to clinical samples was evaluated by studying 50 middle ear fluid (MEF) specimens from children with acute otitis media. Twenty-six of the MEF samples were positive by real-time PCR and the numbers of genome equivalents detected varied from 90 to 88,000/microl in 17 culture-positive samples and from 1 to 1,200/microl in 9 culture-negative samples. The results were compared to culture findings and to results obtained by using agarose gel electrophoresis or Europium-labeled hybridization probes for the detection of amplification products of conventional PCR. The sensitivity and specificity of the real-time PCR assay developed in the present study compared to culture were 100 and 73%, and to conventional PCR with agarose gel and/or TRF detection 93 and 96%, respectively. The real-time PCR assay was found to be rapid, easy to use, and sensitive in detecting and quantifying pneumococci.     

Diagnosis of helicobacter pylori infection by polymerase chain reaction: is it worth it?

The aim of this study was to determine to what degree polymerase chain reaction (PCR) was superior to histology and culture, and whether a noncommercial urease test was of value, in detecting Helicobacter pylori in gastric biopsy specimens. Gastric biopsy specimens from the antrum and corpus of 134 consenting patients were subjected to PCR, targeting the glmM (ureC) gene, histology, culture, and a rapid urease test. PCR detected H. pylori in the biopsy specimens from 59 patients. All methods showed a high degree of sensitivity and specificity, but histology gave 2 false-negatives, and culture and the urease test gave 1 false-negative compared with PCR. PCR of a glmM gene segment was superior to the other methods for the detection of H. pylori infection and was comparable to histology in terms of cost. Nevertheless, in this study, histology and culture were found to be relatively reliable methods for examining gastric biopsy specimens.     

焦磷酸测序法在幽门螺杆菌克拉霉素耐药基因检测及药物疗效评价中的应用

目的本研究建立了幽门螺杆菌(Hp)克拉霉素耐药基因检测的焦磷酸测序方法,在获知耐药情况的同时根据半定量结果初步评估治疗效果。方法通过考察最合适的扩增引物、体系、退火温度建立最优的PCR扩增体系。分别对方法的灵敏度,特异性及一致性进行了评价。通过对44例临床标本的检测,比较本方法与快速尿素酶试验和13 C呼气试验的检出率。结果最优的PCR扩增体系为HiFi体系,最佳退火温度为59.8℃。本方法特异性良好,灵敏度达到100 copies/μL,与Sanger测序结果100.0%一致。36例标本快速尿素酶和焦测序法的检出率分别为48.7%和81.1%,耐药率为10.81%。8例标本13 C呼气试验和焦测序法的检出率分别为75.0%和87.5%,阳性标本检出一致率为100.0%。结论焦测序检测方法具备灵敏度高、快速、半定量的特点,为临床Hp克拉霉素耐药检测及初步疗效评价提供了一种新的方法。     

Detection of influenza virus from throat and pharyngeal swabs with a nested duplex light cycler RT-PCR

A rapid duplex RT-PCR method was developed using the Roche LightCycler technology for detection of influenza type A and influenza type B viruses. Ninety-seven clinical specimens were analyzed using the Lightcycler method compared with conventional viral culture. Thirty-seven specimens (38.1%) were positive by RT-PCR using matrix protein (MP) primers for influenza A or B virus, compared to thirteen culture positive specimens (13.4%). All culture positive specimens were also positive by RT-PCR. A nested PCR reaction using hemagglutination (HA) gene primers confirmed all of the earlier positive PCR reactions. This LightCycler PCR technique was found to be more sensitive than viral culture for influenza virus detection.     

The evaluation of diagnostic methods for the detection of Helicobacter pylori in gastric biopsy specimens

PCR is a rapid, sensitive and accurate method for the specific detection of Helicobacter pylori from gastric biopsy specimens. In our study, 104 gastric tissue specimens from symptomatic adult patients were examined by staining, culture, PCR and nested PCR methods for detection of H. pylori. According to our results, positivity was achieved in 24% (25/104) with Giemsa staining, 34% (36/104) with histopathology, 36% (38/104) with PCR and 41% (43/104) with nested PCR respectively, whereas H. pylori was isolated in only 33% (35/104) of the cultures on the biopsy specimens. Both the sensitivity and the positive predictive value of the nested PCR method were 100%, and both the specificity and negative predictive value were 98%. As a conclusion, our results suggest the nested PCR as a highly valuable method in the detection of H. pylori with a reasonably high sensitivity and specificity.     

HBsAg ELISA-/NAT+的HBV血清学模式和病毒变异分析

目的结合乙型肝炎病毒核酸(HBV DNA)的检测结果,分析血清标本血清学模拟及DNA序列,了解HBV DNA以及HBsAg血型模式之间的关系。方法选自该院54例血液标本作为本研究对象;对标本行HBV DNA提取、PCR扩增处理、PCR产物纯化、DNA测序、HBV基因分型和序列分析,并运用生物信息学软件对测序结果进行分析处理,对比分析基因突变情况。结果 PCR扩增结果,54例标本中40例表现为非常显著的PCR扩增不佳,14例标本PCR产物电泳均能够观察到1 400bp特异性条带;22例为HBsAg ELISA阳性,14例为隐匿性HBV阳性,PCR扩增阳性行基因型检测,B型基因在HBsAg ELISA中占81.82%,C型基因在隐匿性HBV阳性中比例为78.57%,差异具有统计学意义(P0.05);14株隐匿性HBV基因中,6例在S区区域内发生基因序列点突变,其中2例在1个碱基点出现突变,另3例在2个位点的碱基点出现突变;3例HBV基因C型感染者出现基因点突变,2例B基因型感染者出现基因点突变;5例标本在9个位点部位的"a"决定族内碱基点出现突变,A-C的突变较多。结论血液筛查中,检测显示为HBsAg的阴性合格血液,仍可能有隐匿性乙肝感染和窗口期漏检,其病毒株主要为C型,且有变异株,B型病毒株相对较少,但突变株相对较高,可能逃避现有筛查试剂。