肠出血型大肠埃希菌O157：H7 EspF蛋白多克隆抗体的制备和初步纯化

目的制备肠出血型大肠埃希菌（EHEC）O157：H7EspF蛋白多克隆抗体并初步纯化。方法生物信息学方法分析EHECO157：H7EspF蛋白的柔韧性、亲水性、表面可能性及抗原表位,选取1条由15个氨基酸残基组成的多肽为半抗原,在其C端偶联钥孔血蓝蛋白（KLH）,免疫新西兰大白兔制备抗血清。ELISA测定抗血清效价,免疫印迹法鉴定其特异性,辛酸-硫酸铵法初步纯化抗血清,SDS-PAGE检测抗体纯度。结果选择EspF蛋白抗原指数最高的肽段72-TPSRPAPPPPTSGQA-86（0.506）为半抗原,合成抗原多肽经高效液相色谱鉴定,纯度为95.78%,经质谱分析其分子量为1563.76Mr,与目的多肽分子量一致;加强免疫3次后,EHECO157：H7EspF蛋白的抗血清效价达1：2048000;该血清对EHECO157：H7野生株和espF突变株（△espF）的EspF蛋白均有特异性,并经辛酸-硫酸铵法获得一定纯度的抗体。结论成功地制备了效价高、特异性强的EHECO157：H7EspF蛋白多克隆抗体。     

不同遗传谱系肠外大肠埃希菌毒力因子检测

目的 通过检测肠外致病性大肠埃希菌(ExPEC) 30种毒力因子了解其基因的检出情况及遗传谱系分型情况,并初步探讨两者关系,探究ExPEC的分子流行病学特点.方法 210株分离自血液、尿液及其他部位的菌株参与研究,采用多重PCR方法检测临床分离菌株毒力因子基因的存在情况并进行遗传谱系分型,比较菌株遗传谱系分型各组间毒力因子基因的检出情况是否有差异.结果 ExPEC中毒力基因fimH、fyuA、traT、iutA、kpsMTⅡ、PAI的检出率较高;遗传谱系分型表明4组百分比分别为24％、11％、29％和36％.在各组之间进行毒力因子基因检出情况比较显示,毒力因子中有8种仅与B2组联系密切,4种仅与D组联系密切,6种与B2组和D组均有关.结论 ExPEC遗传谱系分型主要属于B2组和D组,ExPEC毒力因子基因检出情况与菌株遗传谱系分型关系密切.     

肠致病性大肠埃希菌感染对靶细胞内钙信号变化的影响

肠致病性大肠埃希菌 (En teropathogenicEscherichiacoli,EPEC) ,经粪 口途径传播 ,是导致婴幼儿腹泻的主要病原体。EPEC的Ⅳ型菌毛是该菌的一个重要的毒力因子 ,在所致的肠道感染中起关键作用。本研究通过比较EPEC野生型菌株E7与无菌毛变异株H5 11感染靶细胞的过程 ,探讨EPEC菌毛对细菌黏附及靶细胞钙信号反应的影响。用ONPG法测定EPEC。将细菌接种于含 0 .2mmol/LIPTG的LB培养基 ,37℃ ,12 0r/min 15h。Hep2细胞种于 96孔板 ,至长成单层贴壁细胞。每孔加入含 10 8/mlEPEC的DMEM培养基 2 0 0 μl,37℃分别孵育 5、10、30…     

肠产毒性大肠埃希菌定植因子研究进展

肠产毒性大肠埃希菌定植因子为近几年发现的一种重要的细菌致病菌毛,它能使大肠杆菌粘附于宿主肠牯膜而不被肠蠕动和肠分泌液所清除,再通过其分泌肠毒素导致婴幼儿和旅行者腹泻。菌毛亚单位的保守区域、抗原决定簇组成、定植因子质粒与毒力编码基因的联系,对研制广谱重组菌毛疫苗具有重要指导意义。本文综述了近几年来有关定植因子菌毛的研究进展,以及与各类肠毒素编码质粒的相关性和疫苗研究概况。     

肠出血性大肠埃希菌 espO基因敲除菌株的构建及其生物学功能初步研究

目的:检测 espO基因敲除对肠出血性大肠埃希菌(EHEC)生物学特性的影响。 方法:自杀质粒pCVD442-Δ espO介导的两步法构建 espO基因敲除菌株(Δ espO),pTrc99a质粒构建 espO基因回补突变株(CΔ esp...     

大肠埃希菌O157：H7Tir基因的生物信息学分析

目的扩增和测序肠出血型大肠埃希菌O157：H7 tir打基因,利用生物信息学预测分析其结构和功能特征．以探讨Tir作为疫苗候选抗原的可能性。方法利用PCR技术扩增fir基因并测序,应用生物信息学网站在线分析工具和vectorNTISUite软件分析Tir蛋白结构和生物学功能,预测B细胞抗原表位。结果该基因全长1674bp,编码558个氨基酸,蛋白质总体亲水性高,有稳定的理化性质。含有3个结构和功能域,两段穿膜结构,多个磷酸化位点,预测20个B细胞线性表位。结论Tir毒力因子是很有前景的疫苗候选抗原,为肠出血型大肠埃希菌O157：H7的疫苗研究提供了理论依据。     

大肠埃希氏菌和肺炎克雷伯氏菌产AmpC酶的检测方法比较

目的：探讨适用于临床微生物常规检测产AmpC酶菌株的方法，为指导临床合理选用抗生素及监控产酶株的流行提供依据。方法：用Tris—EDTA纸片法和聚合酶连反应（PCR）2种试验检测AmpC酶并进行比较。结果：大肠埃希氏菌42株及肺炎克雷伯氏菌18株共60株中，Tris—EDTA纸片法检测出产AmpC酶30％（18株），PCR法为28％（17株）。Tris—EDTA纸片法与PCR法符合率（98．3％），敏感率为100％，特异性为97．8％。结论：Tris—EDTA纸片法简便实用。可作为产AmpC酶菌株的检测方法在基层临床微生物室应用。     

环丙沙星药敏检测与大肠埃希菌gyrA基因突变关系研究

为了解3株3次环丙沙星药敏检测结果不一致的大肠埃希菌gyrA基因突变状况,本文对该3株菌和另3株3次环丙沙星药敏检测均为耐药的大肠埃希菌进行了gyrA基因喹诺酮耐药决定区(QRDR)PCR扩增和产物DNA测序。结果为3株3次环丙沙星药敏检测结果不一致的和另3株3次检测均为耐药的大肠埃希菌均存在gyrA基因第83和第87位氨基酸密码子的突变(TCGTTG,GACAAC)。     

大肠埃希氏菌感染的临床护理

大肠埃希氏菌又名大肠杆菌,主要寄生在大肠内,一般不致病,在一定条件下可引起肠道外感染,如腹膜炎、胆囊炎、尿道感染、腹泻等.该细菌属多重耐药菌,能同时对β-内酰胺类和氨基糖甙类药物产生耐药[1].2008年12月至2009年2月我科发生2例大肠埃希氏菌感染病例,通过精心治疗与护理治愈出院.现将报告如下.     

多重实时荧光定量PCR检测肠出血性大肠杆菌O157:H7

目的 利用多重荧光定量PCR技术,建立一种快速、准确、特异检测肠出血性大肠杆菌O157:H7的定量方法.方法 选取肠出血性大肠杆菌O157:H7编码脂多糖基因(rfbE)和编码鞭毛抗原基因(fliC)作为检测的靶基因,设计引物和TaqMan-MGB探针,探针的5'端分别用FAM和HEX进行荧光标记,3'端标记MGB.优化PCR扩增体系,对多重实时荧光定量PCR方法的特异性、灵敏度、重复性评价,同时进行一定数量临床样本鉴定,与常规方法进行比较.结果 本研究所建立的多重实时荧光定量PCR方法可准确、特异地检测和鉴定肠出血性大肠杆菌O157:H7,能够有效甄别肠出血性大肠杆菌O157:H7与非H7菌株,其他菌株均无阳性结果;该方法的灵敏度可达到10 CFU/ml;定量检测的批间和批内变异系数均小于5%;对66例临床样本进行评价,结果显示15例肠出血性大肠杆菌O157:H7阳性,2例为肠出血性大肠杆菌O157:非H7阳性,其中16例与常规培养法结果符合,符合率达到98.49%.结论 本研究建立的检测肠出血性大肠杆菌O157:H7多重实时荧光定量PCR方法快速,结果准确、可靠,操作简便,为肠出血性大肠杆菌O157:H7的临床诊断、现场流行病学调查和食品安全监测提供了新的鉴定方法.     

Detection of Anaplasma phagocytophilum in animals by real-time polymerase chain reaction

The aim of this study was to detect Anaplasma phagocytophilum in wild and domesticated animals and to identify the phylogenetic relationships of different strains of this bacterium. We adapted six published conventional methods targeting 16S fragments for real-time polymerase chain reaction. Initial screening of samples from 419 animals found 37 Anaplasma positives, later confirmed with several different primers and a TaqMan probe. We also performed DNA quantification and melting curve analysis. The nucleic acid of Anaplasma sp. was detected in a higher percentage of cases in members of the deer family, hares, bank voles and mice (12.5 approximately 15%) than in foxes, boars, cows, and horses (around 4 approximately 6%). We also performed blood analysis of cows, horses, mice, and ticks removed from animals, evaluating the presence of antibodies against granulocytic Anaplasma sp. Finally, we subjected 11 randomly selected PCR amplified products to direct sequencing and we constructed the corresponding phylogenetic tree with respect to the Ehrlichia equi sequence, homologous to the human granulocytic ehrlichiosis agent. Mutual identity of the sequencing ranged from 99% to 100%.     

Standard and real-time multiplex PCR methods for detection of trimethoprim resistance dfr genes in large collections of bacteria

Two multiplex PCR (mPCR) methods were developed to screen large collections of trimethoprim-resistant Escherichia coli isolates for the most prevalent resistance determinants. Five common integron-carried genes (dfrA1, dfrA5, dfrA7, dfrA12 and dfrA17) were selected as PCR targets. Primers and conditions for standard mPCRs and real-time mPCRs were selected and tested. Two protocols using essentially the same primer pairs were established. The standard mPCR protocol also included an internal control targeting the E. coli 16S rRNA gene. Both protocols proved to be sensitive and specific for detection of the five selected genes. Screening of three different collections of clinical urinary and blood isolates (n = 368) with the two multiplex methods revealed that the five dfr genes accounted for 75-86% of trimethoprim resistance. The standard mPCR is useful and accessible for most laboratories, while the real-time mPCR requires additional equipment and expensive reagents, but is very convenient for high-throughput screening of large collections of bacterial isolates.     

Detection of diarrheagenic Escherichia coli by multiplex PCR

Background: Diarrheagenic E.coli (DEC) are an important cause of childhood diarrhea.Identification of DEC strains needs to detect factors that determine the virulence of these organisms. There is not much data regarding the importance of DEC as a cause of diarrhea in children in India.The prevalence of DEC in children belowfive years with and without diarrhea was studied using two multiplex PCR assays. Materials and Methods: Two multiplex polymerase chain reaction assays were used to detect genes of five types of DEC.The targets selected for each category were eae and bfpA (bundle-forming pilus) forEnteropathogenic E.coli (EPEC), hlyA for Enterohemorrhagic E.coli (EHEC), elt and stla for Enterotoxigenic E.coli (ETEC), CVD432 for Enteroaggregative E.coli (EAEC) and ial for Enteroinvasive E.coli (EIEC). Results: In 200 children with diarrhea 52 (26%) DEC infections were found. Among 100 controls 8 (8%) DEC infections were found. EAEC was the most common DEC by multiplex PCR both in cases (26, 13%)and controls (5,5%), followed byEPEC seen in 16% cases and 3% controls. ETEC and EIEC were found in 7 (3.5%) and 3 (1.5%) of the diarrheal cases. EIEC and ETEC were not detected in the control cases. EHEC was not isolated from either the diarrheal or control cases. Conclusion: DEC strains are a significant cause of diarrhea in children. The two Multiplex PCR assays can be used for the detection of DEC in routine diagnostic laboratories. These assays are specific and sensitive for the rapid detection of DEC. EAEC was the most frequent pathotype in the population under study.     

Comparison of a conventional polymerase chain reaction with real-time polymerase chain reaction for the detection of neurotropic viruses in cerebrospinal fluid samples

Purpose: To compare a conventional polymerase chain reaction (PCR) and real-time PCR for the detection of neurotropic DNA viruses. Materials and Methods: A total of 147 cerebrospinal fluid (CSF) samples was collected from patients attending a tertiary care hospital in South India for a period from 2005 to 2008. All these samples were tested using a conventional multiplex/uniplex PCR and a real-time multiplex/uniplex PCR. This technique was used to detect a large number of herpes viruses responsible for central nervous system infections, including HSV-1, HSV-2, VZV, CMV and EBV and the polyoma virus JCV. Results: Overall, in the entire set of samples, the real-time PCR yielded 88 (59.9%) positives and conventional PCR had six (4.1%) positives. Conclusion: Our results suggest that the real-time PCR assay was more sensitive compared with the conventional PCR. The advantage of real-time PCR is that it can be performed much faster than conventional PCR. Real-time PCR is less time-consuming, less labour-intensive and also reduces the chance of contamination as there is no post-amplification procedure. In the entire study population, the major viruses detected using real-time PCR were EBV (34%), HSV-2 (10.8%) and VZV (6.8%).     

大肠埃希氏菌的qnrB基因检测与耐药机制研究

目的探索本地区医院大肠埃希氏菌喹诺酮耐药基因的分布及耐药机制。方法采用PCR扩增与测序、基因初步定位、质粒接合转移实验等方法确定喹诺酮耐药的大肠埃希氏菌qnr的基因类型,以分析研究有关的耐药特点与机制。结果各大肠埃希氏菌株中仅qnrB基因阳性,qnrA、qnrS、qnrC、qnrD、qepA、aac（6’）-Ib-cr基因均阴性;并且qnrB基因包括qnrB2、qnrB5、qnrB9、qnrB16、qnrB18、qnrB19和qnrB31等位基因;在这些qnrB等位基因中,qnrB31与qnrB16、qnrB2与qnrB9同源性较高;各qnrB等位基因分别位于约21.0 kb至28.0 kb长的质粒上。结论在本地区医院存在不同的qnrB等位基因流行;实验菌株的喹诺酮耐药与qnrB等位基因结构中LexA-蛋白结合位点共有序列缺失有关。     

Prevalence of the new,SPI1-like,pathogenicity island ETT2 among Escherichia coli

The new pathogenicity island ETT2 has been identified by PCR and gene probes among various intestinal serovars and pathovars of E. coli, in particular among EHEC/STEC. However, ETT2 was not detected among extra-intestinal and non-pathogenic E. coli strains or other enteric bacteria including various S. enterica serovars. A considerable molecular diversity of ETT2 among various E. coli serovars was found. The occurrence of ETT2 among E. coli is independent of the presence of other virulence properties, e.g. the pathogenicity islands LEE, LPA, or HPI.     

实时荧光PCR检测蓝氏贾第鞭毛虫和隐孢子虫方法的建立

目的建立贾第鞭毛虫和隐孢子虫的基因检测方法。方法根据GenBank中贾第鞭毛虫和隐孢子虫相对保守序列,设计引物及其相应的Taqman探针。通过对引物和探针浓度、Taq酶以及反应条件等优化筛选后,建立检测贾第鞭毛虫和隐孢子虫的荧光PCR方法。结果贾第鞭毛虫和隐孢子虫的检测结果为阳性,对其它DNA样本如日本血吸虫、刚地弓形虫、溶组织内阿米巴、旋毛虫和阴道毛滴虫的检测均为阴性。本法的敏感性高,可检测到10～102copies/μl的DNA浓度。结论建立的基因检测方法,对贾第鞭毛虫和隐孢子虫的检测具有高度的特异性和敏感性,可用于饮用水和临床样本等的快速检测。     

Detection of human immunodeficiency virus-1 by polymerase chain reaction and virus cultivation

Peripheral blood of 57 patients with antibodies to human immunodeficiency virus 1 (HIV-1) and of five HIV-1 seronegative subjects at risk for HIV-1 infection were analysed by polymerase chain reaction (PCR) and virus isolation. The virus was recovered from peripheral blood cells in 89% and from plasma in 75% of the HIV-1 seropositive cases. In contrast, proviral HIV-1 DNA was detected in all HIV-1 seropositive patients by dot blot hybridization of the amplified fragments. The intensities of the dot blot reactions were less pronounced in asymptomatic HIV-1 seropositive individuals than in patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC), suggesting an increase in proviral DNA with advancing disease. Three of five seronegative patients with signs or symptoms suggesting HIV-1 infection, but none of the controls, were positive for HIV-1 DNA by one or two primer pairs. These results show a high sensitivity of the PCR for detecting HIV-1 DNA in patients of all stages of HIV-1 infection. Proviral DNA can also be detected in some individuals without detectable antibodies to the virus. The virus load in peripheral blood, as determined by virus cultivation and PCR, seems to increase with progression of the infection.