随机扩增多态性DNA分析技术及其应用

随机扩增多态性ＤＮＡ分析技术是９０年代发展起来的一项以ＰＣＲ为基础的基因分析方法。本文介绍了该技术的原理、特点、并综述了其在生物种分类鉴定、世系发育及基因分析等生命科学领域中的应用。     

多聚酶链式反应随机引物扩增的DNA多态指模技术在幽门螺杆菌菌株鉴别中的应用

本利用多聚酶链式反应随机引物扩增的DNA(RAPD)多态指模技术研究其在HP菌株鉴别的应用。对47例株HP进行RAPD多态指模分析。结果发现：①40株由不同病人分离的HP菌株有不同的DNA指模；②同一菌株多次培养后其DNA指模无改变；③HP根除疗法后3个月4例、6个月1例(分离的HP菌株分与治疗前相同，提示可能是HP复发；④HP根除疗法后6个月及24个月各1例)与治疗前分离的菌株DNA指模不同．提示可能是HP再感染．结果提示RAPD多态指模技术对HP菌株的鉴别有价值。     

随机扩增多态性DNA基因分型对铜绿假单胞菌克隆菌株的分析

目的 了解铜绿假单胞菌(Pseudomorlas aeruginosa,PA)的基因分型及克隆菌株在医院内感染中所占的比例.方法 通过随机扩增多态性DNA基因分型对69株PA进行克隆菌株的分析.结果 69株PA基因分型36型,有33株菌是同源菌.结论 切断患者之间、患者和护工、患者和护士、医生之间交叉感染可降低PA感染率,加强医院感染控制措施有可能成为降低感染率最重要的关键因素之一.     

随机扩增多态性DNA的研究

随机扩增多态性 DNA(RAPD)是 1990年首次由 William等 [1 ]提出的用于监测 DNA多态性的新型的 PCR技术。它是利用单个的、任意序列的寡聚核苷酸作引物 ,随机地与靶序列完全或部分配对 ,以扩增出特异的 DAN产物。 RAPD方法与传统的 PCR技术比较 ,最显著的优点是 :事先不需要知道目的基因组的任何序列信息 ,便可直接用一任意序列的引物对整个基因组进行分析 ,极大地提高了效率。因其引物序列的任意性 ,没有种属特异性 ,所以不同种属的结果可以相互比较。且灵敏度高 ,能监测出单个碱基的变化 [1 ] 。因此 ,该方法一经提出 ,便迅速得…     

DNA随机扩增多态性分析技术在枸杞道地药材鉴别中的应用

目的:对宁夏和内蒙两个地区的枸杞DNA进行分子标记分析,获得DNA指纹谱。方法:用随机扩增多态DNA(RNAD)标记方法对宁夏和内蒙不同来源地的枸杞进行遗传多样性分析。结果:用 RAPD标记方法可以从DNA分子水平看出宁夏枸杞与内蒙枸杞的差异,结论:本法可为枸杞道地品种鉴别提供依据。     

随机扩增多态性DNA分析在微生物基因分型中的应用研究进展

随机扩增多态性ＤＮＡ分析在微生物基因分型中的应用研究进展郭永建一、微生物基因分型微生物分型对于社区和医院的感染性疾病的流行病学调查和监测、传播途径的了解和阻断、以及发病机理研究，极为重要。传统的表型分型方法，有许多局限性。随着分子生物学的理论和技术高...     

随机扩增DNA多态性技术在A组链球菌分型上的应用

目的 了解A组乙型溶血性链球菌的基因分型状况。方法 采用随机扩增多态性DNA技术(Random amplified polymorphic DNA，RAPD)，对1988～1994年度．从广东城市和农村，1993～1994年度，从湖北、吉林9～11岁学龄儿童分离的179株GAS进行RAPD分析。结果 ①同一T型的菌株有特征性条带，但有些亚型带型间差异较大：②不同T型间可有相同的带型；③分型率比T分型高，达到96．6％；④有主导RAPD型菌株流行的情况存在。结论 RAPD方法对GAS分型及流行病学中有一定的应用价值，可以区别同一血清型不同地区来源的GAS。     

4种硬蜱的随机扩增多态性DNA分析

蝉分布广，种类繁多，可以传播多种人兽共患病，在医学上有着重要的意义。蜱的分类与疾病的关系也为许多科学家所重视。近年来，随着科学技术的不断发展，许多新的技术手段也逐渐引入了节肢动物的分类中。1990年，Williams等首先使用单一的10碱基任意DNA序列作为引物扩增DNA，以观察各物种间基因的多态性。他们证实，这种基因的多态性可用于基因的连锁分析，所得结果符合孟德尔定律[1]。随机扩增多态性DNA（RandomAmplifiedPoly－morphicDNA，PAPD）技术由于其简捷、灵敏、对材料要求不高、成本低等优点，从它一问世，就备受人们的…     

四种蝇的随机序列扩增多态性分析

ＲＡＰＤ技术是在１９９０年后发展起来的一项分子生物学手段，它被越来越广泛地应用在昆虫分类方面。它有许多优越性，如简便、快捷、灵敏度强、经济、对材料要求不高等。更重要的是，它可以从分子的角度出发揭示ＤＮＡ的多态性，从而把物种区分开来。本文以家蝇，丝光绿蝇，尾黑麻绳和夏厕蝇为对象，用３４种１０ｈｐ引物对上述四种蝇的ＤＮＡ进行了地毯式的扩增，以筛选能够用于种类区别的引物。我们的结果显示，引物ＯＰＨ－８可以作为科间的鉴别依据，另有１４种引物可用于蝇科内各属间或种间的分类依据。     

金黄色葡萄球菌随机扩增多态性DNA指纹图与药敏谱对医院感染管理的指导意义

目的 探讨金黄色葡萄球菌(SA)药敏谱型对医院感染管理的指导意义。方法 通过随机扩增多态性DNA(RAPD)技术分型76株SA，并归纳出药敏谱型与RAPD型的对应关系。结果 药敏谱型35种，RAPD分型50种。二者呈单一对应关系的有22种，呈一对二关系的有12种，呈一对三关系的有1种。结论 依据SA药敏谱型可对SA进行分型，从而指导医院感染管理。     

H pylori recurrence after successful eradication

Recurrence of H pylori after eradication is rare in developed countries and more frequent in developing countries. Recrudescence （recolonization of the same strain within 12 mo after eradication） rather than reinfection （colonization with a new strain, more than 12 mo after eradication） is considered to be responsible for most of the cases. This observation was confirmed only in developed countries, while in developing countries a recent meta-analysis demonstrated a high rate of reinfection. The proportion of H pylori annual recurrence was 2.67% and 13.00% in developed and developing countries, respectively. Nested meta-analysis （only cases with a longer follow-up and a negative 13CUBT a year after eradication） revealed annual recurrence rate of 1.45% [relative risk （RR）, 0.54] and 12.00% （RR, 0.92） in developed and developing countries, respectively. These findings support the notion that in developed countries many cases of recurrence are due to recrudescence within the first year after eradication, with a 46% drop in the recurrence rate after the first year post eradication, while in developing countries reinfection is more pronounced, and continue at the same rate since eradication. A different approach for follow-up after H pylori eradication is probably needed in patients of developing countries, since reinfection is highly prevalent.     

Detection of H. pylori antibody profile in serum by protein array

AIM: To detect multiple H pylori antibodies in serum samples of individuals who carryH pylori by protein array. METHODS: Recombinant H pylori antigens, urease B subunit (UreB), vacuolating toxin A (VacA) and cytotoxin associated gene A protein (CagA), were prepared and immobilized in matrixes on nitrocellulose membrane by robotics to bind the specific immunoglobulin G (IgG) antibodies in serum. Staphylococcus protein A (SPA) labeled by colloid gold was used to integrate the immuno-complex and gave red color signal. The scanner based on charge-coupled device (CCD) could collect the image signal and convert it into digital signal. RESULTS: When human IgG was printed on the membrane in increasing concentrations and incubated with immunogold, a linear dose response curve was obtained and the detection limit for IgG was about 0.025 ng. The cutoff values, which were defined as the mean grey level plus 3 times of standard deviation, were 27.183, 28.546 and 27.402, for anti-UreB IgG, anti-CagA IgG and anti-VacA IgG, respectively, as 400 human serum samples with negative H pylori antibodies were detected by the protein array. When 180 serum samples from patients in hospital were employed for detection of IgG against UreB, CagA and VacA, the sensitivity of the protein array was 93.4%, 95.4%, 96.0%, and the specificity was 94.8%, 94.4% and 97.5%, respectively, as compared with the results obtained by ELISA. The assay also showed high reproducibility, uniformity and stability, and the results were available within 30 min. CONCLUSION: The protein array is a very practical method for rapid detection of multiple antibodies in serum samples. It is especially useful for large scale epidemiological investigation of the infection of H pylori.     

Helicobacter pylori neutrophil activating protein as target for new drugs against H. pylori inflammation

Helicobacter pylori(H.pylori) infection is among the most common human infections and the major risk factor for peptic ulcer disease and gastric cancer.With-in this work we present the implication of C-terminal region of H.pylori neutrophil activating protein in the stimulation of neutrophil activation as well as the evi-dence that the C-terminal region of H.pylori activating protein is indispensable for neutrophil adhesion to endothelial cells,a step necessary to H.pylori inflammation.In addition we show t...     

幽门螺杆菌感染与上胃肠道疾病

1982年Marshall和Mareen首次从慢性活动性胃炎患者的胃粘膜中分离出为幽门螺杆菌(Hp)。本文概括了Hp与上胃肠道疾病的关系，并评估其感染的治疗。现已确认Hp与4种上胃肠道疾病密切相关：(1)慢性胃炎；(2)消化性溃疡病；(3)胃癌；(4)胃粘膜相关淋巴样组织(MALT)淋巴瘤。Hp是慢性胃炎的主要病因，与消化性溃疡病的发生密切相关，Hp感染增加了胃腺癌发生的危险性，而且也涉及到胃MALT淋巴瘤发生的致病机理。Hp感染的治疗是以PPI、铋制剂以及RBC为基础的三联疗法，当三联疗法失败时则推荐四联疗法。四联疗法是传统的三联疗法(铋剂为基础的三联疗法)十PPI组成。     

Identification of H. pylori in Saliva by a Nested PCR Assay Derived from a Newly Cloned DNA Probe

A novel probe was developed from genomic DNA ofHelicobacter pylori ATCC type strain 43629. Ithybridized with all 73 H. pylori clinical isolatestested but not with any of 183 non-H. pylori DNAs in dot blot hybridization. Typing tests revealed 41different HaeIII-digestion patterns from 57 H. pyloristrains tested. Based on the sequence of the probe, anested PCR was developed that detected as little as 2 fg of H. pylori DNA or approximatelyequivalent to one cell. No PCR products were amplifiedfrom any of 21 non-H. pylori strains tested. Using thisnested PCR, H. pylori DNA was detected in 33 of 45 (73%) saliva samples collected from patientswith gastric H. pylori infection. These data suggestthat the probe is useful for typing H. pylori and thatthe nested PCR is a valuable tool for detecting H. pylori DNA in saliva.     

Peroxiredoxin I protects gastric mucosa from oxidative injury induced by H. pylori infection

Background and Aim: Helicobacter pylori (H. pylori) infection enhances the production of reactive oxygen species and peroxynitrite, thereby resulting in oxidative tissue damage. In this study, we examined the role of peroxiredoxin I (Prx I), a stress‐induced antioxidant enzyme, in protecting gastric mucosa from H. pylori‐induced gastric mucosal injury. Methods: Wild type (Prx I^+/+) and Prx I‐deficient type (Prx I^–/–) mice were maintained for 2 to 12 months with or without infection of H. pylori, Sydney strain‐1. Gastric mucosal expression of Prx I was assessed by immunoblot analysis and immunohistochemistry. The degree of gastritis was evaluated by the updated Sydney system and by mucosal levels of inflammatory cytokines (MIP‐2, IL‐1β, and TNF‐α). Oxidative DNA injury and apoptosis were analyzed by mucosal level of 8‐hydroxy‐2′‐deoxyguanosine, and the number of apoptotic cells stained with a single‐stranded DNA antibody, respectively. Results: H. pylori infection upregulated gastric mucosal Prx I expression in the Prx I^+/+ but not the Prx I^–/– mice. H. pylori infection also induced more severe gastritis and a more prominent increase in MIP level, more marked oxidative DNA injury, and apoptosis in the Prx I^–/– than the Prx I^+/+ mice. In the absence of H. pylori infection, no changes were demonstrated in gastric mucosa in either the Prx I^+/+ or the Prx I^?/? mice. Conclusion: These data suggest that H. pylori infection upregulates gastric mucosal Prx I expression, and further, that Prx I plays an important role in gastric mucosal protection against oxidative injury induced by H. pylori infection.     

幽门螺杆菌感染对Fas/FasL表达的影响在胃癌发生中的作用

目的：观察幽门螺杆菌（Hp）及其不同毒力株（cagA阳性与cagA阴性株）感染对胃黏膜上皮细胞Fas/FasL表达的影响，进而探讨胃癌的发生机制。方法：胃镜下取胃窦黏膜标本，将研究对象按病理结果分为黏膜萎缩组，黏膜萎缩伴轻度不典型增生组，黏膜萎缩伴中度不典型增生组，胃腺癌组，黏膜大致正常组为对照组，再根据Hp感染情况为分Hp阳性组与阴性组，并将Hp阳性组进一步成cagA阳性组及阴性组，共9组80例。以快速尿素酶试验，PCR及组织学第三种方法检测Hp，用PCR方法对Hp进行分型。用免疫组化法检测Fas、FasL等表达情况。结果Hp感染率为60．0％，cagA阳性率为90．47％，非腺癌病人Hp阳性组Fas/FasL表达明显高于Hp阴性组（P＜0．05），腺癌组Fas/FasL表达明显高于大致正常组及黏膜萎缩，黏膜萎缩伴轻度不典型增生，黏萎缩伴中度不典型增生组（P＜0．01）。cagA阳性组Fas/FasL表达与cagA阴性组差异无显著性（P＞0．05）。结论：幽门螺杆菌感染后早期即黏 萎缩阶段已出现Fas、FasL等的表达增加，随细胞凋亡的增加，黏膜上皮细胞萎缩加重，细胞DNA不稳定性增加，并出现不典型增生加重， Fas、FasL的表达随之增强，一旦肿瘤细胞形成，Fas/FasL表达进一步增加，形成局部免疫豁免区，导致肿瘤的浸润生长。cagA阳性的菌株在促成肿瘤的发生过程中无明显作用。     

幽门螺杆菌感染对胃上皮细胞增殖影响的实验研究

目的在幽门螺杆菌(H.pylori)长期感染蒙古沙土鼠腺胃模型基础上,探讨H.pylori感染诱致胃癌发生的可能机制.方法采用H.pylori国际标准菌株NCTC11637灌喂蒙古沙土鼠,建立H.pylori长期感染动物模型;用免疫组化及原位杂交方法检测H.pylori感染致胃上皮细胞增殖的改变.结果成功建立了H.pylori长期感染蒙古沙土鼠腺胃模型,其胃粘膜的组织学变化显示,H.pylori感染可致正常胃粘膜→慢性胃炎→萎缩→肠化生→异型增生的发展过程.H.pylori感染对胃上皮细胞增殖的影响:H.pylori感染能引起胃窦上皮细胞增殖增加(P<0.05);随着H.pylori感染时间的增加,EGF mRNA及EGFR mRNA的阳性信号表达呈逐渐增加的趋势(P<0.05) H.pylori定植致胃窦上皮细胞增殖的异常,对正常胃窦粘膜向不典型增生进展的过程起重要作用:EGF及EGFR在mRNA水平的异常表达可能是H.pylori定植致胃窦上皮细胞增殖异常的重要原因。     

幽门螺杆菌感染对胃上皮细胞凋亡影响的实验研究

目的在H.pylori长期感染蒙古沙土鼠腺胃模型基础上,探讨H.pylori感染诱致胃癌发生的可能机制.方法采用H.pylori国际标准菌株NCTCli637灌喂蒙古沙土鼠,建立H.pylori长期感染动物模型:用末端脱氧核苷酸转移酶介导的dUTP切口末端标记法(TUNEL)及原位杂交方法检测H.pyiori感染致胃粘膜上皮细胞凋亡的变化。结果成功建立了H.pylori长期感染蒙古沙土鼠腺胃模型,其胃粘膜的组织学变化显示,H.pylori感染可致正常胃粘膜→慢性胃炎→萎缩→肠化生→异型增生的发展过程。H.pylori感染对胃上皮细胞凋亡的影响:接种H.pylori后25周内能引起胃窦粘膜上皮细胞凋亡增加(P<0.05),H.pylori感染45周时,与25周时相比,胃窦粘膜上皮细胞凋亡指数呈逐渐递减的趋势,65周时甚至明显低于正常值(P<0.05):FAS及FASL原位杂交检测结果显示随着H.pylori感染时间的延长,炎症及病变程度的加重,FAS及FASL mRNA的阳性信号表达有逐渐增加的趋势(R<0.05)。结论 H.pylori走植致胃窦粘膜上皮细胞凋亡的异常,对正常胃窦粘膜向不典型增生进展的过程起重要作用;FAS和FASL在mtLNA水平的异常表达可能是H.pylori定植致胃窦粘膜上皮细胞凋亡异常的重要原因.     

Natural maternal transmission of H. pylori in Mongolian gerbils

INTRODUCTION H pylori is a gram-negative, spiral-shaped, microaerophilic bacterium that infects the human gastric mucosa[1]. Chronic infection is thought to be associated with chronic active gastritis, peptic ulcer and gastric malignancies, such as mucosa…