espP基因在O157∶H7大肠埃希菌分离株中的分布

目的了解espP基因在中国部分地区产志贺毒素O157大肠埃希菌分离株中的分布情况及特点。方法根据O157∶H7的溶血素基因hlyA和细胞外分泌蛋白基因espP设计特异性引物,对113株O157大肠埃希菌分离株进行PCR检测。结果 hlyA的阳性率为99.1%,espP的阳性率为72.5%,且只在有大质粒pO157的菌株中检测到espP。结论 espP存在于携带pO157大质粒的O157∶H7大肠埃希菌中,其分布与菌株来源、志贺毒素携带情况等没有相关性。     

肠出血性大肠埃希菌O157 ∶ H7质粒 pO157 上毒力因子的研究进展

肠出血性大肠埃希菌O157 ∶ H7是一种重要的致病菌,可以引起不同程度人类疾病。目前已知的O157 ∶ H7的致病因子有3个,志贺毒素、粘附抹平效应毒力岛和特异性质粒pO157。其中pO157在致病过程中的作用并不十分清楚。本文结合近年来的研究进展,介绍了pO157上与细菌致病性有关的毒力因子。     

多重PCR检测肠出血性大肠埃希菌O157：H7

目的 建立一种快速、特异的检测肠出血性大肠埃希菌O157:H7菌株的多重PCR方法。 方法 针对O157:H7菌株的O157、H7抗原特异基因rfbE O157、fliC H7以及stx1、stx2、eaeA和hlyA四种毒力基因设计相应引物,在同一扩增体系中进行PCR反应,通过优化多重PCR 反应条件和循环参数,建立检测O157:H7菌株的多重PCR方法,并测定其特异性和灵敏度。 结果 6 对特异性引物各自扩增相应的基因片段,检测结果与常规PCR 获得的结果一致。细菌纯培养物的检测灵敏度为1.33×10⁴ CFU。 结论 该多重PCR方法能在一次检测中同时反映待测菌株是否为肠出血性大肠埃希菌O157:H7及其携带毒力基因的情况,可为O157:H7大肠埃希菌感染的诊断及流行病学调查提供一种简便、快速的检测手段。     

鉴定大肠埃希菌O157:H7的表型特征探讨

目的探讨大肠埃希菌O157:H7特有的表型特征并应用于鉴定。方法用4-甲基伞型酮-β-D-葡萄糖醛酸苷(MUG)和山梨醇试验鉴别3株大肠埃希菌O157:H7标准菌株以及大肠埃希菌ATCC 25922(ATCC 25922)、侵袭性大肠埃希菌(EIEC)、产肠毒素大肠埃希菌(ETEC)、肠致病性大肠埃希菌(EPEC)和肠聚集性大肠埃希菌(EAEC),并用全自动微生物鉴定系统VITEK 2-compact等自动化仪器及血清学做进一步鉴定,对健康体检者粪便中检出的大肠埃希菌也进行了鉴定。结果 3株大肠埃希菌O157:H7的MUG和山梨醇均阴性,EPEC O111的MUG阳性、山梨醇阴性,其他大肠埃希菌的MUG和山梨醇均阳性。来自粪便的357株大肠埃希菌中,检出MUG阴性或山梨醇阴性及MUG和山梨醇均为阴性的大肠埃希菌95株。大肠埃希菌O157:H7被VITEK 2-Compact明确筛查出来。仅大肠埃希菌O157:H7与O157血清凝集,试验的其他大肠埃希菌及粪便中的95株大肠埃希菌全部不与O157血清凝集。ATB半自动微生物仪及基质辅助激光解吸-飞行时间质谱仪(VITEK MS)不能鉴定大肠埃希菌O157:H7。结论 MUG和山梨醇发酵试验联合应用,对大肠埃希菌O157:H7有较高的筛查准确率,效果优于其他方法,但必须经血清学鉴定。     

江苏省建湖地区大肠埃希菌O157:H7毒力基因分析

目的对检测出的17份肠出血性大肠埃希菌0157：H7阳性菌株进行毒力基因分析。方法收集江苏省建湖地区2008年5月～9月监测出的17株肠出血性大肠埃希菌0157：H7阳性菌株,利用单一和多重PCR法检测不同来源菌株0157抗原编码（rfbE）、H7鞭毛抗原编码（fliC）、志贺样毒素（stx1和stx2）、溶血素（hly）和黏附抹平因子（eaeA）。结果通过血清学方法确定的17株肠出血性大肠埃希菌0157：H7阳性菌株,再通过PCR法检测,其中15株大肠埃希菌rfbE和fliC基因检测为阳性,确认为EHEC O157：H7,其中9株菌株扩增出全部毒力基因,5株菌株扩增出除stxl外其它全部毒力基因,1株仅检出stx2,hly毒力基因;2株大肠埃希菌rfbE基因检测阳性、fliC基因检测为阴性,确认为O157：H7-大肠埃希菌,无毒力基因检出。结论该地区存在O157：H7大肠埃希菌强毒株污染,从流行病学角度看具潜在的流行性危险,当地疾控部门应加强O157：H7的综合监测。     

一种鉴别与大肠埃希菌O157血清凝集的弗氏枸橼酸杆菌的PCR方法

目的利用基于O抗原翻转酶wzx基因的PCR方法,鉴别与大肠埃希菌O157血清凝集的弗氏枸橼酸杆菌和O157大肠埃希菌。方法用微生物鉴定仪对细菌进行生化鉴定,与O157血清凝集,同时扩增wzx基因。结果25株与大肠埃希菌O157血清凝集的弗氏枸橼酸杆菌及1株不与大肠埃希菌O157血清凝集的弗氏枸橼酸杆菌wzx基因扩增为阳性,8株与O157血清不凝集的弗氏枸橼酸杆菌wzx基因扩增为阴性,20株O157∶H7大肠埃希菌wzx基因扩增为阴性。结论对于可与O157血清发生强凝集的弗氏枸橼酸杆菌,基于O抗原翻转酶wzx基因的PCR方法是一种有效的快速鉴别方法。     

动物性食品中大肠埃希菌O157：H7检测分析

目的 了解本县动物性食品中大肠埃希菌O157:H7及带菌情况,为预防O157:H7引起的爆发流行提供依据.方法 采用常规培养法对部分动物性食品样品进行大肠埃希菌O157:H7培养分离和鉴定.结果 235份样品中检出4份大肠埃希菌O157:H7,检出率为1.7%.结论 生鲜牛奶和生猪肉中检出大肠埃希菌O157:H7,应引起高度重视.     

免疫PCR技术检测肠出血型大肠埃希菌O157:H7

目的 通过检测肠出血型大肠埃希菌O157:H7菌株,确定免疫PCR技术的检测灵敏度和特异性,探讨该方法用于检测食品和临床标本的可行性。 方法 免疫PCR技术检测O157:H7标准菌株和自食品、临床标本分离的菌株。 结果 免疫PCR最少可检测101/ml O157:H7细菌,临床和食品分离的O157:H7菌株检测结果均为阳性,而非O157:H7菌株检测结果均为阴性。 结论 免疫PCR技术具有较高的检测灵敏度、特异性及操作简便等特点,可作为食品和临床标本检测O157:H7方法。     

2008-2010年浙江省衢州市动物粪便O157∶H7监测及其意义

目的 动态了解浙江省衢州市家禽、家畜肠出血性大肠埃希菌O157∶H7带菌情况和菌型分布特征,以制定相应的防制对策。 方法 6、7月肠道传染病高发季节,采集动物粪便标本,对O157∶H7菌株进行分离培养,并用PCR方法检测其毒力基因。 结果 共采集动物粪便标本914份,检出O157∶H7菌2株,总带菌率为0.22%,其中羊、牛各检出1株,带菌率分别为0.62%和0.71%。经PCR检测,其中1株携带stx2+ hly+ eaeA毒力因子,1株hly+eaeA阳性,stx1均为阴性。 结论 衢州市再次分离到带有毒力基因的肠出血性大肠埃希菌O157∶H7,对人群健康构成了威胁,应加强O157∶H7的综合监测。     

基于夹心法的DNA生物传感器用于检测大肠埃希菌O157：H7 stx2基因

目的构建DNA生物传感器用于大肠埃希菌O157:H7的快速检测。方法基于双探针夹心法,结合酶标生物电催化反应技术和计时电流的电化学法检测大肠埃希菌O157:H7的stx2基因。结果在最佳反应条件下,DNA探针能较好地固定于金电极表面;探针标记链酶亲合素后,检测信号明显增强。DNA生物传感器具有良好的特异性,其值可达6.25×10-8mol/L。结论构建的DNA生物传感器特异性和灵敏度高,可为大肠埃希菌O157:H7的快速检测提供新的方法。     

Development of PCR primers based on a fragment from randomly amplified polymorphic DNA for the detection of Escherichia coli O157:H7/NM

Serotype O157:H7 of EHEC is by far the most prevalent serotype associated with haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS). Although PCR methods aimed on the detection of genes associated with the pathogenicity of Escherichia coli O157:H7 have been reported, tests allowing the direct identification of this serotype are rare. In this study, we used RAPD-PCR tests to analyze strains of E. coli O157:H7 serotype, strains of non-pathogenic E. coli, and strains of other pathotypes, including enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), and enteroaggregation E. coli (EAggEC). One RAPD fragment co-shared by serotype O157:H7 strains was observed when 10-mer primer termed as OPQ3 was used. After sequencing this fragment, three primers were designed and combined to form two PCR primer pairs. These two primer pairs were highly specific to the strains belonging to E. coli O157:H7/NM (non-motile).     

Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes

A multiplex PCR assay was developed which allowed the simultaneous detection of five trait genes or virulence markers in enterohemorrhagic Escherichia coli (EHEC) serotypes. A primer pair, designed to detect a single base-pair mutation in the uidA gene, is specific only for the prototypic EHEC of O157:H7 serotype and its toxigenic, non-motile variants. In a similar way, primers to the eaeA gene of the gamma-intimin derivative specifically detects strains in the EHEC 1 clonal group, which consists mostly of O157:H7 and some O55:H7 serotypes. The other three primer pairs, specific for stx1, stx2 and both variants of ehxA genes, will detect the presence of these virulence genes in all EHEC serotypes. Analysis of 34 strains, including various serotypes of EHEC, Shiga toxin-producing E. coli and enteropathogenic E. coli, confirmed that the multiplex PCR assay detected the presence of these genes in a manner consistent with the known genotype of each respective strains.     

Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes

A multiplex PCR assay was developed which allowed the simultaneous detection of five trait genes or virulence markers in enterohemorrhagic Escherichia coli (EHEC) serotypes. A primer pair, designed to detect a single base-pair mutation in the uidA gene, is specific only for the prototypic EHEC of O157:H7 serotype and its toxigenic, non-motile variants. In a similar way, primers to the eaeA gene of the γ-intimin derivative specifically detects strains in the EHEC 1 clonal group, which consists mostly of O157:H7 and some O55:H7 serotypes. The other three primer pairs, specific for stx1, stx2 and both variants of ehxA genes, will detect the presence of these virulence genes in all EHEC serotypes. Analysis of 34 strains, including various serotypes of EHEC, Shiga toxin-producing E. coli and enteropathogenic E. coli, confirmed that the multiplex PCR assay detected the presence of these genes in a manner consistent with the known genotype of each respective strains.     

Optimization of a 3'-minor groove binder-DNA probe targeting the uidA gene for rapid identification of Escherichia coli O157:H7 using real-time PCR

Enterohemorrhagic Escherichia coli are harmful human pathogens capable of causing bloody diarrhea and vomiting. An important serotype commonly associated with human illness is the E. coli O157:H7 serotype. Unlike other real-time polymerase chain reaction (PCR) methods for identifying E. coli O157:H7, this study describes the development and optimization of a real-time PCR method targeting a conserved point mutation at +93 in the uidA (gusA) gene that is unique to O157:H7, distinguishing it from non-O157:H7 serotypes. A TET-labeled Minor Groove Binder (MGB) DNA probe was designed for use in a 5' nuclease PCR assay. Using a panel of two E. coli O157:H7 strains, three E. coli non-O157:H7 strains, and one non-E. coli species, the assay was optimized for the specific detection of the E. coli O157:H7 strains. Optimal conditions were identified at high anneal/extend temperatures, low magnesium concentrations, and low probe concentrations, resulting in correct identification of E. coli O157:H7 and non-O157:H7 strains. The improved specificity of MGB probes for single base pair mismatches such as the +93 uidA mutation provides a novel approach towards rapid identification of E. coli O157:H7.     

浙江省检出带毒力基因的人源性肠出血性大肠杆菌O157:H7

目的了解肠出血性大肠杆菌O157:H7在浙江省人群中的感染情况和菌型分布特征,制定相应的防治对策。方法5～10月份肠道传染病高发季节,在浙江省各地(市)肠道门诊采集腹泻病人粪便,对O157:H7菌株进行分离培养,并用PCR方法检测其毒力基因。结果1998年开始至今的人群监测中,共检出2株无毒力的肠出血性大肠杆菌O157:H7。PCR毒力基因检测,SLT2和Hly阳性,SLT1阴性。结论与以往不同,此次分离到带有毒力基因的人源性肠出血性大肠杆菌O157:H7,说明浙江省O157:H7在菌型特征方面发生了变化,对人群健康构成了更大的威胁。     

安徽省114株EHECO157∶H7菌株毒力因子基因的初步分析

目的　对安徽省近几年分离的肠出血性大肠杆菌O157∶H7(EHECO157∶H7)菌株的毒力因子基因进行实验室检测 ,以分析和研究肠出血性大肠杆菌O157∶H7的毒力特征和致病特性。方法　采用聚合酶链反应(PCR)检测EHECO157∶H7的两种重要的毒力因子志贺样毒素 (SLT)和溶血素 (hly)的特异性基因片段 ,并辅以血清学试验、生化试验进行菌株的生物学特性鉴定。结果和结论　检测结果显示 ,有 63.2%的EHECO157∶H7菌株携带SLT、hly基因 ,动物源的菌株以家禽较高 ,家畜次之 ,3株源自腹泻病人的菌株毒力因子基因携带率为33.3% ,说明分离的EHECO157∶H7菌株具有较强的致病性 ;114株菌株中,有 14.0%的菌株在 24h内发酵山梨醇 ,发酵山梨醇菌株的毒力因子基因携带率 18.8% ,显著低于不发酵菌株的 70.4% ,显示出山梨醇发酵特性变异与毒力因子基因缺失之间具有一定的关联性。     

PCR primers targeting the 16S rRNA gene for the specific detection of streptomycetes

Streptomycetes are filamentous actinobacteria commonly found in soil and biotechnically important, but they also have adverse effects on human health. In this work, two primer pairs, StrepB/StrepE and StrepB/StrepF combined with Bst YI restriction endonuclease digestion, targeting the 16S rRNA gene of streptomycetes were designed. The specificity of the primers was determined by polymerase chain reaction (PCR) amplification from Streptomyces strains and near relatives. All streptomycetes tested positive and non-streptomycetes were not amplified except three strains that, however, gave Bst YI restriction endonuclease digestion results distinct from streptomycetes. Moreover, both primer pairs gave an amplification product of the expected size only when Streptomyces VTT E-99-1334 DNA was present in the template DNA mixture isolated from six bacterial and three fungal strains. The primers were further successfully used to amplify from DNA isolated from two soil and two building material samples. The 40 sequenced amplification products obtained with the primer pair StrepB/StrepE showed greater than 96.1% similarity to streptomycete 16S rRNA sequences. Seventy PCR amplification products obtained with the primers StrepB/StrepF were analysed by sequencing and restriction analysis. All 54 PCR products having >95.7% similarity to streptomycete sequences were cleaved with Bst YI. No false-positive results were achieved. Both primer sets proved to be specific for streptomycetes, and applicable for the detection of streptomycetes in environmental samples.     

昆明市首次检出肠出血性大肠杆菌O157∶H7

为了解昆明市是否存在肠出血性大肠杆菌O15 7∶H7及流行情况 ,了解蔬菜、食物、水等的污染情况 ,2 0 0 1年 10月采集昆明市鸡、鸭、牛 3个养殖场、西式快餐店 2个和市区 9个农贸市场的鸡、鸭、牛粪 ,生牛奶 ,生、熟肉馅 ,生蔬菜 ,生、熟猪、牛肉 ,海产品 ,以及猪、鱼、鸡内脏等 14种共 15 5份样品。按照大肠杆菌O15 7∶H7的常规分离鉴定方法和分子生物学和血清学试验鉴定 ,结果共发现 19株大肠杆菌O15 7∶H7,并且 19株菌株均含有志贺样毒素VT2的致病基因。其中从蔬菜 (33份 )分离到 9株、鸡内脏 (9份 )分离到 3株、生猪肠 (17份 )和卤猪肉 (12份 )各分离到 2株 ,发干菜 (6份 )、鱼内脏 (6份 )、海产品 (6份 )各分离到 1株大肠杆菌O15 7∶H7。食品中检出阳性菌株数之多在全国尚属少见。特别是蔬菜 ,其检出O15 7∶H7大肠杆菌阳性数 ,占总检出数的 4 3 37% ,与国内其它地区有所不同。昆明市存在发生肠出血性大肠杆菌O15 7∶H7感染暴发或流行的潜在危险.