豆类及其制品中大肠杆菌O157和O104的双重real-time PCR检测方法

目的建立一种快速检测豆类及其制品中肠出血性大肠杆菌O157、O104的双重real-time PCR方法。方法分别选取大肠杆菌O157的特异性基因rfbE和大肠杆菌O104的特异性基因wzy作为靶基因,设计相应的特异性引物探针,建立双重real-time PCR反应体系,并对反应体系及引物浓度等反应条件进行优化。结果最终确定反应体系为:以大肠杆菌O157、大肠杆菌O104菌株的DNA按照1∶3比例混合作为模板,模板DNA 2μL,Master Mix 12.5μL,上游引物(10μmol/L)各1μL,下游引物(10μmol/L)各1μL,探针(10μmol/L)各0.5μL,灭菌水补齐至25μL。实现了同一次处理样品在同一反应体系中同时检测肠出血性大肠杆菌的两种血清型,检测灵敏度的下限可达102CFU/m L(g)。结论该方法特异性好,对其他常见病原菌无扩增,灵敏度高、快速、简便,为食源性致病菌的检测提供了理想手段。     

食品样品中大肠杆菌O157:H7复合PCR检测方法的研究

根据大肠杆菌O15 7:H7特异性基因fliC ,rfbE ,SLTI与SLTII设计四对引物 ,建立了复合PCR方法 ,扩增产物长度分别为为 5 6 0bp ,6 78bp ,2 10bp与 4 84bp。该方法可以一步检测出O15 7与H7特异性抗原基因 ,并可同时检测该菌产生SLT毒素的类型 ,与 71株试验菌株的基因型完全配合 ,是一种特异、高效的检测方法。该方法适用于牛奶样品中大肠杆菌O15 7:H7的检测 ,经过增菌步骤检测限可达 0 1cfu ml     

12株疑似O157：H7大肠菌PER鉴定研究

目的：对12株疑似O157：H7大肠菌采用PCR法进行鉴定。方法：利用单一PCR和多重聚合酶链反应（mPCR）检测不同来源菌株志贺样毒素（stx1和stx2）、溶血素（hly）、粘附抹平因子（eaeA）、β-葡糖醛酸糖苷酶（uidA）、O157抗原编码（血E）、H7鞭毛抗原编码（niC）基因。结果：4株大肠菌rfbE和fliC基因检测为阳性，确认为EHEC O157：H7，其中1株菌株扩增出全部毒力基因，另3株菌株扩增出除stx1外其它全部毒力基因；2株大肠菌rfbE基因检测阳性，确认为O157：H7-大肠菌；其它均为非O157：H7其它大肠菌。结论：PCR技术的应用能对可疑O157：117大肠菌进行有效鉴定与分析，应成为今后病原学鉴定的主要技术手段。     

17株产H_2S致泻大肠埃希菌的微生物学研究

目的对水源水和腹泻病人标本中检测到的17株产H2S致泻大肠埃希菌进行一系列微生物学实验研究,为进一步了解该菌的流行病学特点以及临床诊断提供实验依据。方法自1999年10月～2005年8月,从腹泻病人标本和水源水中分离的产H2S致泻大肠埃希菌,经形态学、培养特性、生化学、血清学等微生物学鉴定,并且对菌株进一步进行了药敏试验和噬菌体裂解试验。结果在腹泻、食物中毒病人标本和水源水中分离到17株产H2S致泻大肠埃希菌,经血清学分型后,其中4株是EPEC O128：K67,ETEC O25：K19和ETEC O16：K15分别为3株,占58.8%。结论产H2S致泻大肠埃希菌与大肠埃希菌有密切的亲缘关系,是变异的致泻大肠埃希菌,在致泻大肠埃菌中占有较高的比例。致泻大肠埃希菌产H2S生化变异提示我们在注意生化反应典型的致病菌分离与鉴定时,还应注意生化变异菌株的检测和鉴定,防止该菌造成新的腹泻病的传播和蔓延。     

大肠杆菌O157多克隆抗体及食品中双抗ELISA测定方法的研究

本研究获得了抗肠出血性大肠杆菌O157:H7的多克隆抗体 ,建立了一种适宜食品样品检测的双抗ELISA检测方法。该方法对纯培养菌液检出限为 10 3 ～ 10 4 cfu ml;只对O157菌株有特异性反应 ,对非O157菌株无交叉反应 ;经过增菌 ,鸡肉与牛奶染菌样品中的大肠杆菌O157的检出限均为 0 1cfu g(cfu ml)。     

A combined AFLP-multiplex PCR assay for molecular typing of Escherichia coli strains using variable bacterial interspersed mosaic elements

The original method for molecular typing of E. coli strains was developed using the polymorphism in chromosomal sequences of bacterial interspersed mosaic elements (BIMEs) detected by multiplex PCR and analysed by AFLP assay. The applicability of the method in the epidemiology of E. coli was tested on a group of 524 strains of human and veterinary origin. In the studied group 18 different genotypes were detected. Significant differences were found in the frequencies of the genotypes among various groups of strains, suggesting the method could be a promising tool in the epidemiology of E. coli.     

12株疑似O157:H7大肠菌PCR鉴定研究

目的:对12株疑似O157:H7大肠菌采用PCR法进行鉴定。方法:利用单一PCR和多重聚合酶链反应(mPCR)检测不同来源菌株志贺样毒素(stx1和stx2)、溶血素(hly)、粘附抹平因子(eaeA)、β-葡糖醛酸糖苷酶(u idA)、O157抗原编码(rfbE)、H7鞭毛抗原编码(fliC)基因。结果:4株大肠菌rfbE和fliC基因检测为阳性,确认为EHEC O157:H7,其中1株菌株扩增出全部毒力基因,另3株菌株扩增出除stx1外其它全部毒力基因;2株大肠菌rfbE基因检测阳性,确认为O157:H7-大肠菌;其它均为非O157:H7其它大肠菌。结论:PCR技术的应用能对可疑O157:H7大肠菌进行有效鉴定与分析,应成为今后病原学鉴定的主要技术手段。     

Construction of non-toxic Escherichia coli and Vibrio cholerae strains expressing high and immunogenic levels of enterotoxigenic E. coli colonization factor I fimbriae

To express high quantities of colonization factor antigen I (CFA/I) derived from enterotoxigenic Escherichia coli (ETEC) for use in ETEC vaccines, the entire CFA/I operon consisting of four genes (cfa-A, -B, -C, -E) was cloned into plasmid expression vectors that could be maintained either with or without antibiotic selection. Expression from the powerful tac promoter was under the control of the lacIq repressor present on the plasmids. Fimbriae were expressed on the surface of both a non-toxigenic E. coli K12 strain and a non-toxigenic strain of Vibrio cholerae following induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). It was found that the recombinant E. coli strains expressed up to 16-fold higher levels of CFA/I fimbriae compared to a reference strain which had previously been shown to be among the highest natural producers of the CFA/I fimbriae among tested wild type ETEC strains. Oral immunization with formalin-killed recombinant E. coli bacteria over-expressing CFA/I induced significantly higher serum IgA and IgG+M antibodies responses compared to the reference strain. Oral immunization with formalin-killed recombinant V. cholerae bacteria also induce strong CFA/I-specific serum IgA and IgG+M responses. We conclude that our constructs may be useful as candidate strains in an oral killed CF-ETEC vaccine.     

Studies into the incidence of carriers of enteropathogenic E. coli.

Enteropathogenic Escherichia coli (EPEC) is frequently found in asymptomatic carriers. When checked over a period of two and a half months at fortnightly intervals, nine out of 20 children at a child care centre in Jamaica showed EPEC of three different serotypes in faecal smears. Four stool specimens were found to be positive in another 45 children, controlled routinely on admission to a district hospital in Jamica. None of either group had diarrhoea at the time of collection. These results confirm that serotypes should not be equated with pathogenicity. Also, current methods of detecting enterotoxin are too time-consuming and impractical for non-specialized laboratories. The place of antibiotics in EPEC diarrhoea is criticized. Initial control measures should centre on fluid and electrolyte replacement irrespective of the aetiology of the diarrhoea.