检测大肠杆菌(Escherichia coli) O157:H7免疫磁珠的制备、保存与应用

【背景】大肠杆菌(Escherichia coli) O157:H7是导致肠出血性大肠杆菌食源性疾病暴发的主要血清型,免疫磁珠(Immunomagnetic beads,IMBS)在E. coli O157的检测中发挥着重要作用,而免疫磁珠的稳定性、特异性、广谱性等性能指标关系着在实际应用中的使用效果。【目的】制备高效、稳定且具有广谱性的免疫磁珠,联合分子检测技术如环介导恒温扩增 (Loop-mediated isothermal amplification,LAMP)技术、PCR等,提高目标菌的检出率。【方法】采用新型的磁珠活化剂MIX&GO制备E. coli O157免疫磁珠,并进行广谱性以及特异性检测;针对6种试剂牛血清白蛋白(Bovine serum albumin,BSA)、酪蛋白(Casein)、海藻糖(Trehalose)、聚乙烯吡咯烷酮(Polyvinyl pyrrolidone,PVP)、抗坏血酸(Vitamin C)和防腐剂ProClin 300,利用正交试验L18(37)优化免疫磁珠保存液组分;采用IMBS-LAMP、IMBS-PCR、IMBS-生化、菌液-LAMP、菌液-PCR、显色平板-生化鉴定6种方式对20份生猪肉样品进行检测。【结果】利用MIX&GO活化剂制备的免疫磁珠捕获率最高达到81.5%±1.3%;免疫磁珠保存液最优配方为：牛血清白蛋白15.0 g/L,酪蛋白10.0 g/L,海藻糖10.0 g/L,PVP 2.0 g/L,抗坏血酸5.0 g/L,ProClin 300 2.5 g/L,保存6个月后免疫磁珠捕获率为75.5%;在20份生猪肉样品的检测中,自制磁珠和商品化磁珠与LAMP联用均检出9例阳性样品;IMBS-LAMP在6种检测方式中具有最高的检测灵敏度,但检出的样品会因磁珠抗体的差异而有所不同。【结论】与商品化磁珠相比,实验制备的免疫磁珠具有良好的特异性和广谱性,免疫磁珠-LAMP联用提高了目标菌的检出率,是一种高灵敏度、具有应用前景的检测方法。     

变性高效液相色谱检测沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌

应用多重PCR 反应(multiplex PCR,mPCR)结合变性高效液相色谱(denaturing high-performance liquid chromatography,DHPLC)技术建立食品中沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌O157:H7的快速检测方法.以编码沙门氏菌的fimY基因、编码空肠弯曲菌的gyrA基因和编码肠出血性大肠杆菌O157:H7的rfbE基因为靶基因,选择3对引物,建立并优化了快速鉴别沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌O157:H7的多重PCR体系,扩增产物分别为284、159和499 bp,并验证了该多重PCR具有特异性.沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌O157:H7标准菌株稀释成不同梯度,做灵敏度检测.试验结果表明该方法有很好的特异性,且灵敏度高,检测限可达到:沙门氏菌1.5 CFU/ml、空肠弯曲菌15 CFU/ml、肠出血性大肠杆菌O157:H7 15 CFU/ml.在随机采集的226份冷冻鸡肉类样品中,检出了7份样品为沙门氏菌阳性、10份为空肠弯曲菌阳性、1份为肠出血性大肠杆菌O157:H7阳性.研究建立的多重PCR-DHPLC方法可特异、灵敏地实现对沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌O157:H7的快速检测.     

肠出血性大肠杆菌（EHEC）O157：H7紧密粘附素免疫保护性片段（Intimin-C）的克隆表达纯化及部分生物学活性研究

克隆表达并纯化肠出血性大肠杆菌(EHEC)O157:H7紧密粘附素免疫保护性片段(Intimin-C),并对其部分生物学活性进行研究.     

肠出血性大肠杆菌O157：H7监测及分析

为了了解长春地区动物和人感染肠出血性大肠杆菌O157H7状况,建立流行病学监测网.采集长春市动物养殖场动物粪便和腹泻病人便样进行监测.结果在牛粪和鸡粪中共检出2株O157H7大肠杆菌.可见,在长春地区存在肠出血性大肠杆菌O157H7菌潜在污染的威胁,需要加强监测力度.     

肠出血性大肠杆菌(O157：H7)的特异性荧光探针检测方法的建立

目的:建立一种real-time PCR,快速准确检测肠出血性大肠杆菌O157:H7。方法:以肠出血性大肠杆菌0157:H7 rfbE为待检靶基因,设计一对引物和一条Taqman探针,探针5’端用FAM基团标记,3’端用TAMRA标记。通过重组质粒的构建,建立并优化了大肠杆菌0157:H7的荧光定量PCR检测方法。结果:在人工污染样本无需富集的情况下,检测的最低DNA浓度是10拷贝/反应(3CFU/mL);特异性检测实验中,0157菌株检测结果均为rfbE阳性,而非0157:H7菌株检测结果均为阴性;重复性实验中,批内、批间变异系数均小于3%。结论:实验结果显示此荧光定量PCR方法特异性、灵敏度高,重复性好,可对分离的可疑大肠杆菌0157:H7菌株进行快速鉴定。     

大肠杆菌O157:H7致病因子及其保护性免疫研究现状

大肠杆菌O157:H7是肠出血型大肠杆菌(EHEC)的主要病原血清型,它可产生特殊的粘附因子粘附靶细胞,产生Vero毒素和肠溶血素毒力因子.近几年对大肠杆菌O157:H7的毒力因子有了深入的了解,对致病机理作了一些探讨,用实验动物对保护性免疫进行了研究.本文对近几年来0157:H7大肠杆菌的致病因子及其主要的保护性免疫的研究作一简要综述.     

肠出血性大肠杆菌Ⅲ型分泌系统ATP水解酶escN基因突变株的构建

目的:利用λ噬菌体的Red重组系统敲除肠出血性大肠杆菌O157∶H7的Ⅲ型分泌系统ATP水解酶Esc N,构建大肠杆菌esc N基因缺失突变株。方法:以O157∶H7为模板,PCR扩增目的基因两侧的同源臂序列,分别酶切连接于p UC19-kan质粒上,PCR获得中间嵌合卡那霉素抗性基因(带有FRT位点)的同源线性片段,利用质粒p KD46和p CP20介导的重组技术敲除esc N基因,并去除抗性标记;PCR及测序验证目的基因缺失后,测定缺失株及野生菌株的生长曲线。结果:敲除了肠出血性大肠杆菌O157∶H7的esc N基因,突变株与野生株的生长曲线相近。结论:构建了Ⅲ型分泌系统缺陷菌株,为进一步研究Ⅲ型分泌系统因子在肠出血性大肠杆菌致病过程中的作用奠定了基础。     

肠出血性大肠杆菌毒力相关基因突变株的构建

目的:利用Red重组系统敲除肠出血性大肠杆菌O157∶H7的毒力基因espA、espB、espD,构建3株突变株。方法:以肠出血性大肠杆菌O157∶H7为模板,PCR扩增基因两翼的同源序列;将PCR产物插入pEASY-T1载体并测序,将测序正确的上、下游同源序列分步酶切,构建于pUC19-kan质粒上,经PCR获得两端同源序列中间嵌合卡那霉素抗性基因标记的线性片段,利用质粒pKD46介导的重组技术,敲除espA、espB、espD基因,之后转入pCP20质粒以去除抗性标记,最后测定突变株及野生菌株的生长曲线。结果:敲除了肠出血性大肠杆菌O157∶H7的毒力基因espA、es pB、espD,获得3株突变株,突变株与野生株的生长曲线相近。结论:为进一步研究espA、espB、espD基因在肠出血性大肠杆菌O157∶H7致病过程中的作用奠定了基础。     

肠出血性大肠杆菌Ⅲ型分泌系统及其转位效应器蛋白质

肠出血性大肠杆菌(enterohemorrhagic Escherichia coli,EHEC)0157:H7是一种重要的肠道病原微生物,感染后可引发多种疾病,严重者可导致死亡.EHEC O157:H7通过Ⅲ型分泌系统(TTSS)将其转位效应器蛋白质转位至宿主细胞,经一系列的信号传导过程介导与宿主细胞的"黏附与擦拭"(attaching and effacing,A/E)损伤.对EHEC0157:H7 Ⅲ型分泌系统及其转位效应器蛋白质进行研究,可使我们进一步认识EHEC以及引起A/E损伤的病原菌的致病机理,丰富有关Ⅲ型分泌系统和致病岛的知识.     

大肠杆菌O157:H7的sRNA伴侣蛋白Ufq的基因克隆、表达及纯化

目的:克隆、表达并纯化肠出血性大肠杆菌(EHEC)O157:H7的sRNA伴侣蛋白Hfq.方法:利用PCR方法从EHEC O157:H7基因组中扩增出基因hfq,并插入含6xHis标签序列的原核表达载体pET28a(+)的多克隆位点中,构建重组表达质粒pET28a(+)-hfq,以重组质粒转化大肠杆菌BL21(DE3)...     

Prevalence of Escherichia coli O157:H7 in industrial minced beef

AIMS: The lack of baseline data on the prevalence of Escherichia coli O157:H7 in retail minced beef in France prompted this survey of industrial minced beef production. METHODS AND RESULTS: An automated enzyme-linked fluorescence immunoassay (ELFA), the VIDAS E. coli O157 method, was used to detect E. coli O157 in industrial minced beef samples. Confirmation of samples positive according to the ELFA was performed using an automated immunoconcentration (ICE) system, VIDAS ICE, which allows the selective capture and release of target organisms. The ICE was followed by culture on cefixime tellurite sorbitol MacConkey agar and a chromogenic medium, O157:H7 ID. Of the 3450 minced beef samples tested, 175 samples were positive with the ELFA method and, of these, four were confirmed by the ICE method. They were identified as sorbitol-negative, O157-positive, H7-positive, mobile, verotoxin-producing E. coli. CONCLUSIONS: The prevalence of E. coli O157:H7 in industrial French minced beef was 0.12%, consistent with many other reports. SIGNIFICANCE AND IMPACT OF THE STUDY: The low infective dose of E. coli O157:H7 presents a major threat. The main means of combating this organism are thermal destruction and good food hygiene covering activities on-farm, in the abattoir and in minced beef industries.     

Comparison of culture and immunoassay for detection of Escherichia coli O157 in raw minced meat and hamburger

Verocytotoxin-producing Escherichia coli O157 (VTEC) is an important food-borne pathogen of humans. The serious complications of VTEC infection and the established reservoir of VTEC in cattle used for mass food production are a public health concern. In this study 500 samples of hamburger and minced meat were examined for presence of E. coli O157. For E. coli detection, Tryptic Soy Broth supplemented (with novobiocin and bile salts) and Sorbitol Mc Conkey agar were used; an automated rapid enzyme linked fluorescent immunoassay (VIDAS E. coli O157) was also evaluated. E. coli O157 was found in 5 samples of hamburger, 2 strains were found to be positive for verocytotoxin production on Vero cells.     

Development of an immunomagnetic bead-immunoliposome fluorescence assay for rapid detection of Escherichia coli O157:H7 in aqueous samples and comparison of the assay with a standard microbiological method

The objective of this study was to develop and optimize a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30- to 100-ml samples followed by incubation of the filter membranes or pellet with anti-E. coli O157:H7 immunomagnetic beads in growth medium specific for E. coli O157:H7. The resulting E. coli O157:H7-immunomagnetic bead complexes were isolated by magnetic separation, washed, and incubated with sulforhodamine B-containing immunoliposomes specific for E. coli O157:H7; the final immunomagnetic bead-E. coli O157:H7-immunoliposome complexes were again isolated by magnetic separation, washed, and lysed with a n-octyl-beta-d-glucopyranoside to release sulforhodamine B. The final protocol took less than 8 h to complete and had a detection limit of less than 1 CFU of E. coli O157:H7 per ml in various aqueous matrices, including apple juice and cider. To validate the protocol at an independent facility, 100-ml samples of groundwater with and without E. coli O157:H7 (15 CFU) were analyzed by a public health laboratory using the optimized protocol and a standard microbiological method. While the IMB/IL fluorescence assay was able to identify E. coli O157:H7-containing samples with 100% accuracy, the standard microbiological method was unable to distinguish E. coli O157:H7-spiked samples from negative controls without further extensive workup. These results demonstrate the feasibility of using immunomagnetic beads in combination with sulforhodamine B-encapsulating immunoliposomes for the rapid detection of E. coli O157:H7 in aqueous samples.     

The optimization of isolation media used in immunomagnetic separation methods for the detection of Escherichia coli O157 in foods

AIMS: To compare media used in immunomagnetic separation (IMS) techniques for the isolation of Escherichia coli O157 from food. METHODS AND RESULTS: Foods, both naturally contaminated and spiked, with low numbers (< 1 g(-1)) of stressed E. coli O157 were enriched in media based on buffered peptone water (BPW), tryptone soya and EC broths incubated at 30, 37, 40 and 42 degrees C. Following immunomagnetic separation, beads were plated on a range of selective agars. CONCLUSION: BPW supplemented with vancomycin (8 mg l(-1)) incubated at 42 degrees C, followed by IMS and subsequent plating of immunobeads onto cefixime tellurite sorbitol MacConkey agar plus either Rainbow or CHROMagar agars, proved optimum for the recovery of spiked, stressed E. coli O157 in minced beef, cheese, apple juice and pepperoni. The same protocol was optimum for recovery from naturally-contaminated minced beef and cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimum protocol would increase isolation rates of E. coli O157 from foods.     

Detection of Escherichia coli O157 in French food samples using an immunomagnetic separation method and the VIDASTME. coli O157

C. VERNOZY-ROZAND, C. MAZUY, S. RAY-GUENIOT, S. BOUTRAND-LOEï, A. MEYRAND AND y. richard. 1997. Two commercially available screening methods, an automated enzyme-linked fluorescent immunoassay (VIDAS^TM E. coli O157) and an immunomagnetic separation followed by culture onto cefixime tellurite sorbitol MacConkey agar (CT-SMAC), were compared for detection of Escherichia coli O157 in naturally and artificially contaminated food samples. A total of 250 naturally contaminated food samples, including raw milk cheeses, poultry, raw sausages and ground beef retail samples, were examined. Four poultry, one raw sausage and one ground beef sample were found to be positive for E. coli O157 by both methods. Of the six positive samples, five were shown to contain sorbitol-positive, O157-positive, H7-negative, motile and non-verotoxin-producing E. coli .     

大肠杆菌O157显色培养基检测效果的评价

【目的】评价显色培养基对大肠杆菌O157:H7(Escherichia coli O157:H7)的检测效果。【方法】本实验室研制的大肠杆菌O157显色培养基(HKM),与国外梅理埃、科玛嘉及国内厂家的同类产品及传统培养基CT-SMAC作比较,对相关菌株以及污染样品和实际样品进行对比测试。【结果】实验室研制的HKM大肠杆菌O157显色培养基与科玛嘉同类产品在特异性、灵敏度及检测效果方面均无明显差异,均优于梅里埃、国内厂家产品及CT-SMAC。【结论】HKM大肠杆菌O157显色培养基具有高检出率及高特异性的特点,具有较好的应用价值和前景。     

A solid phase fluorescent capillary immunoassay for the detection of Escherichia coli O157:H7 in ground beef and apple cider

A solid phase fluorescence-based immunoassay was developed for the detection of Escherichia coli O157:H7 using an antigen down competition format. A soft glass capillary tube served as the solid support, to which heat-killed E. coli O157:H7 were adsorbed. Polyclonal anti- E. coli O157:H7 antibody, conjugated with biotin, was used and the bound antigen-antibody complex was detected using avidin molecules labelled with Cy5, a fluorescent cyanine dye. Any E. coli O157:H7 in the sample would compete with the formation of this complex, reducing fluorescence. This assay was tested for sensitivity with spiked ground beef and apple cider samples. The minimum detectable number of cells present in the initial inoculum was calculated to be approximately 1 colony-forming unit (cfu) per 10g of ground beef when samples were enriched in modified EC broth for 7 h at 37°C. The minimum detectable number of cells for the apple cider samples was calculated to be ∼0.5 cfu ml^-1 The E. coli cells in the cider samples were captured with immunomagnetic beads, incubated for 7 h in the enrichment broth, and detected with the solid phase fluorescence immunoassay.     

Fluctuations in the occurrence of Escherichia coli O157:H7 on a Norwegian farm*

AIM: To describe the distribution of Escherichia coli O157:H7 on a sporadically positive dairy farm and on possible contact farms over a one-year period. METHODS AND RESULTS: Environmental and faecal samples from all animals at the farm, and faecal samples from animals at contact farms were analysed for E. coli O157:H7 by immunomagnetic separation methods or VIDAS.Confirmed isolates were tested for cytotoxicity in the Vero cell assay and typed by PFGE. Escherichia coli O157:H7 (stx2 and eae) of the same PFGE type were isolated from cattle, sheep, hens and environmental samples at variable levels during summer and fall 2002, but were not detected in 2003. CONCLUSIONS: Escherichia coli O157:H7 had a widespread distribution on the farm investigated, but the original source of contamination could not be identified. The occurrence of this bacterium on the farm did not result in any detectable increase in gastrointestinal disease in the associated population. SIGNIFICANCE AND IMPACT OF THE STUDY: Despite a low endemic level of E. coli O157:H7 in the Norwegian cattle population, the growth and spread of this potentially important bacterium may occur.     

Immunomagnetic separation of Escherichia coli O26, O111 and O157 from vegetables

AIMS: Raw fruits and vegetables have been increasingly associated with human infections caused by Shiga toxin-producing Escherichia coli. This study evaluates the isolation and detection of E. coli O26, O111 and O157 from vegetable samples using immunomagnetic particles. METHODS AND RESULTS: Standard cultivation and immunomagnetic separation (IMS) procedures were compared. It was found that immunomagnetic particles could efficiently concentrate E. coli cells, detecting significantly more bacteria than with standard cultivation procedures. CONCLUSION: Bacteria were detected in 93-100% of the inoculated samples using the IMS procedure, but only 36-93% samples tested by standard cultivation procedures were found to be positive. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that E. coli O26, O111 and O157 immunomagnetic particles can be a very useful and efficient tool for the detection of E. coli strains in raw vegetables, and could probably be used with samples of animal origin.