基因芯片检测肠出血性大肠埃希菌O157∶H7

目的设计和制备快速、特异、灵敏的检测大肠埃希菌O157∶H7基因芯片。方法选择O157∶H7特异的rfbE、fliC、SLT1和SLT2基因,设计引物和探针,并制备检测芯片,通过两次聚合酶链反应(PCR)扩增,制备荧光标记的靶序列,并与芯片进行杂交,检测O157∶H7菌株和非O157病原体。结果O157∶H7菌株在采用单一和多重PCR两种方法制备的荧光标记靶序列与芯片杂交,均在芯片相应探针处出现阳性信号,非O157杂交结果均为阴性;芯片检测灵敏度比PCR检测高。结论基因芯片可以快速、灵敏、特异地检测O157∶H7,为建立快速灵敏的检测细菌病原体和鉴别诊断的自动分析系统提供了新方法。     

大肠埃希菌O157出血性肠炎

肠出血大肠埃希菌 (Ｅ .Ｃｏｌｉ)主要包括Ｏ157:Ｈ7和Ｏ2 6 :Ｈ11,其中Ｏ157:Ｈ7是出血性肠炎的主要致病菌 ,由Ｒｉｌｅｙ等人于 1982年在美国一次出血性肠炎爆发流行时最先分离出。现对大肠埃希菌Ｏ157出血性肠炎作简要综述。　　大肠埃希菌Ｏ157出血性肠炎的病原学Ｅ .ＣｏｌｉＯ157革兰染色阴性 ,直杆状 ,约 1.0 μｍ×2 .0 μｍ大小。典型的Ｅ .ＣｏｌｉＯ157不发酵山梨醇 ,缺乏 β 葡萄糖醛酸酶 (ＧＵＤ)的活性。在 ｐＨ 2 .5～ 3.0、温度 37℃时能耐受 5ｈ而不失去活性 ,在低温下存活时间较长 ,不耐热 ,在 75℃时 1ｍｉｎ即失去活性[…     

肠出血性大肠埃希菌O157∶H7外膜蛋白A的表达、鉴定和纯化

目的 构建肠出血性大肠埃希菌O157∶H7外膜蛋白A基因重组质粒,研究其在大肠埃希菌中的表达情况. 方法 用RT-PCR法从肠出血性大肠埃希菌O157∶H7菌株克隆OmpA基因,构建重组质粒pET-30a (+)-OmpA,经双酶切及基因测序鉴定后在E.coli BL21(DE3)原核表达系统中诱导OmpA的表达,通过蛋白质电泳技术检测蛋白的表达,用Western blot法测定和分析免疫反应性. 结果 获得的目的 基因为1 041 bp,与预期值相符,测序结果与GenBank公布序列的同源性达100%,重组质粒pET-30a (+)-OmpA在原核系统下可表达OmpA, Western blot分析His-Tag单抗能与OmpA(His-Tag)发生免疫反应. 结论 OmpA重组质粒构建成功,表达产物具有抗原性.     

广西肠出血性大肠埃希菌O157监测研究

目的了解广西人和动物中EHECO157的流行强度与规律,为卫生决策提供依据。方法采集腹泻病人和家禽家畜粪便、苍蝇及生熟肉类食品标本,经mEC肉汤增菌、O157快诊金卡筛选和免疫磁珠富集后,接种CHROMagarO157显色培养基,最后进行生化和血清学鉴定。结果2007-2008年,广西共采集各类标本3964份,从牛粪、猪粪、鸡粪和苍蝇标本中检出O157菌株22株,检出率分别为4.35%、1.27%、0.70%和1.84%,其它标本均未检出。22株O157菌株中有18株O157∶H7、4株O157:H-,分布于4-11月。结论广西家禽家畜和苍蝇均有较高的EHECO157带菌率,存在人感染甚至发生人间暴发流行的危险,应继续加强监测并采取适当措施以降低家禽家畜带菌率。     

肠出血性大肠埃希菌O157：H7外膜蛋白A的表达、鉴定和纯化

目的构建肠出血性大肠埃希菌O157：H7外膜蛋白A基因重组质粒，研究其在大肠埃希菌中的表达情况。方法用RT-PCR法从肠出血性大肠埃希菌O157：H7菌株克隆OmpA基因，构建重组质粒pET-30a（＋）-OmpA，经双酶切及基因测序鉴定后在E．coliBL21（DE3）原核表达系统中诱导OmpA的表达，通过蛋白质电泳技术检测蛋白的表达，用Western blot法测定和分析免疫反应性。结果获得的目的基因为1041bp，与预期值相符，测序结果与GenBank公布序列的同源性达100％，重组质粒pET-30a（＋）-OmpA在原核系统下可表达OmpA，Westernblot分析His-Tag单抗能与OmpA（His-Tag）发生免疫反应。结论OmpA重组质粒构建成功，表达产物具有抗原性。     

上海市浦东新区肠出血性大肠埃希菌O157：H7的监测研究

目的 了解上海浦东新区腹泻病人、宿主动物肠出血性大肠埃希杆菌(E. coli )O157:H7携带情况以及市售食品的污染状况,为疾病防治工作提供决策依据. 方法 于2003～2006年的5～10月,在设置的监测点与超市采集家禽和腹泻病人粪便及禽畜肉制品;应用免疫磁珠富集,山梨醇麦康凯平板和Chromager O157显色平板分离培养E. coli O157:H7,生化和血清学反应进行鉴定. 结果 E. coli O157:H7阳性率分别为:腹泻病人粪便0.24%,鸡粪2.95%,鸭粪3.83%,家畜肉15.05%,家禽肉2.25%.检出的67株E. coli O157:H7中,仅1株来自腹泻病人的E. coli O157:H7具有毒力基因. 结论 浦东新区腹泻病人、宿主动物及其肉类食品中存在E. coli O157:H7感染或污染,存在E. coli O157:H7感染流行的潜在危险.     

肠出血性大肠埃希菌(O157:H7)的基因同源性的分析

目的 对江苏省徐州地区O157：H7的病原学进行分析。方法 采用聚合酶链反应对O157：H7菌株毒力基因谱进行检测，同时用脉冲凝胶电泳(PFGE)和随机扩增多态性DNA(RAPD)方法对O157：H7菌株的同源性分析比较。结果 流行地区分离的O157：H7菌株，100％携带Hly、eaeA基因，95．35％携带SLT2基因，11．63％携带SLT1基因。脉冲凝胶电泳图谱表明流行地区分离的O157：H7菌株与日本分离的O157：H7菌株有明显差异，为不相关菌株；与国内标准菌株882364为近似型(相似，但不相同)。流行地区患者分离菌株与外环境家畜家禽粪便及昆虫肠道分离菌株的脉冲凝胶电泳图谱完全相同。结论 携带O157：H7菌株的家畜家禽可能是导致疫情发生的传染源。脉冲凝胶电泳方法用于O157：H7病原学分析，对流行病学研究有重要意义。随机扩增多态性DNA方法用于O157：H7病原学分析，技术简便、省时。     

肠出血性大肠埃希菌O157：H7感染分子流行病学方法研究进展

肠出血性大肠埃希菌O157∶H7严重危害着人类的健康,已成为世界范围内的公共卫生问题。随着分子生物学的发展,质粒图谱分析,脉冲场凝胶电泳分型、随机扩增多态性分析、限制性片段长度多态性分析、扩增片段长度多态性分析、多位点可变数量串联重复序列分析、多位点测序分型等技术被先后应用于O157∶H7感染的分子流行病学研究,本文对其研究进展进行了简要的综述。     

大肠埃希菌O157：H7

1982年，在Michigan和Oregon因进食污染的汉堡包发生的47名血性腹泻患者粪便中第一次分离出E．coliO157:H7，回顾性研究发现它与1975年一名自限性的急性血性腹泻患者粪便培养的大肠埃希菌为一类，并且1973～1982年间发生的3千多份出血性肠炎E．coli培养中均见O157：H7血清型[1]．它可产生一种与志贺氏毒素（Shigatokin，ST）极相似的毒素，故称之为类志贺氏毒素。又因它对培养的Vero细胞具毒性作用，故又称之为"Vero毒素"。该菌因引起出血性腹泻而得名肠出血性大肠埃希氏苗（enterohemorrhagicE．coli，EHEC）。近年来，更多的研究…     

肠出血性大肠埃希菌O157:H7Ⅱ型志贺毒素的纯化及功能鉴定

目的 亲和层析纯化肠出血性大肠埃希菌(EHEC)O157:H7Ⅱ型志贺毒素,并鉴定其生物学功能.方法 用抗-Ⅱ型志贺毒素分子A亚单位的抗体S1D8耦联至柱填料Sepharose 4B,制备亲和层析柱.纯化EHEC O157:H7菌体分泌的毒素分子,分别用聚丙烯酰胺凝胶电泳(SDSPAGE)和Western印迹法鉴定毒素分子纯度和特异度,将纯化毒素倍比稀释,观察其对Vero细胞和小鼠的毒性作用,计算其对细胞半数致死量(CD50)和对小鼠的全数致死量(LD100);观察抗毒素血清对小鼠进行毒素攻击的保护作用.结果 通过亲和层析从EHEC O157:H7培养物中成功纯化Ⅱ型志贺毒素.SDS-PAGE显示,其A、B亚单位的相对分子质量分别为32 000和7 500,纯化的毒素分子可分别与Ⅱ型志贺毒素A、B亚单位特异性的单抗结合;对Vero细胞和小鼠均存在致死作用,其CD50和LD100分别为20 ng/L和5 ng,小鼠体内抗毒素血清对毒素可有效中和.结论 成功纯化Ⅱ型志贺毒素,并证实其在细胞和动物模型中的毒性作用.     

Antagonistic interaction between Clostridium butyricum and enterohemorrhagic Escherichia coli O157:H7

Antagonistic interaction between Clostridium butyricum strain MIYAIRI 588 and enterohemorrhagic Esherichia coli (EHEC) strain O157:H7 006 was examined using streptomycin-treated SPF mice and germ free mice. All SPF mice pretreated with streptomycin were colonized with EHEC O157:H7. On the other hand, only 20% of the SPF mice pretreated with streptomycin and C. butyricum were colonized with EHEC O157:H7. In addition, germ free mice died within 4-7 days after infection with EHEC O157:H7. In contrast, all gnotobiotic mice mono-associated with C. butyricum survived after the challenge with EHEC O157:H7. Both the number of EHEC and the amounts of shiga-like cytotoxin (SLT, type 1 and type 2) in fecal contents of gnotobiotic mice treated with C. butyricum were less than those of mice infected with only EHEC O157:H7. In conclusion, the probiotic bacterium, C. butyricum strain MIYAIRI 588, has a preventive effect against EHEC O157:H7 infection.     

出血性大肠杆菌O157菌壳的制备研究

目的利用温度控制噬菌体PhiX174裂解基因E的表达,制备出血性大肠杆菌O157∶H7菌壳,鉴定其裂解效率并观察其形态。方法利用PCR技术扩增得到噬菌体PhiX174裂解基因E并克隆到质粒pBV220中,将重组质粒导入出血性大肠杆菌O157∶H7。重组菌株O157∶H7(pBV220::E)培养温度从28℃突升至42℃,每20min检测菌液OD600值,绘制重组菌株生长曲线。体外菌落计数计算裂解效率,并通过电镜观察菌壳结构。结果获得了PhiX174裂解基因E全长基因及重组质粒,构建了大肠杆菌的重组菌株。重组菌菌液OD600值在温度突升至42℃后40min后开始显著下降,80min后OD600值趋于平稳。细菌的裂解效率为98.4%。电子显微镜观察显示,经诱导后,O157∶H7细菌内容物可以通过细菌表面的孔道流出,从而形成菌壳。结论本研究成功制备了大肠杆菌O157∶H7菌壳,为将来进一步研究O157菌壳的特性奠定了基础。     

Escherichia coli O157:H7

E. coli O157:H7 can cause potentially lethal illness in hosts of all ages. These patients often are evaluated and treated by gastroenterologists. The treating physician should administer adequate hydration, usually parenterally, and avoid the use of antibiotics and antimotility agents. The physician needs to notify immediately the appropriate public health authorities of the diagnosis and to ensure that the isolate is recovered by the microbiologist and forwarded for molecular linkage analyses.     

Antibacterial effects of cacao mass on enterohemorrhagic Escherichia coli O157:H7]

The antimicrobial activities of aqueous cacao mass extract against enterohemorrhagic Escherichia coli (EHEC) O157:H7 006 strain were studied. Hot water extract of cacao mass (cocoa extract) was shown to inhibit the growth of EHEC O157:H7 006 strain in PBS or CAYE medium. In addition, the production of verotoxins (types 1 and 2) of EHEC O157:H7 006 strain was significantly inhibited by 8.0% cocoa extract. The cocoa extract did not neutralize the cytotoxity of verotoxins, but had inhibitory effect on adhesion of verotoxins to the target Vero cells. These results demonstrate that cacao mass has antimicrobial effects on EHEC O157:H7.     

Characterization of recombinant antibodies developed for capturing enterohemorrhagic Escherichia coli O157:H7

Escherichia coli O157:H7, an emerging cause of food-borne disease with the occurrence of an estimated 20,000 illnesses and 250 deaths each year in the United States, has now been reported from several countries worldwide. Infections with this bacteria, which follows the ingestion of contaminated food by humans, causes bloody diarrhea, hemolytic uremic syndrome (HUS), and renal disease, that can have serious health implications. The source of food contamination is usually associated with animals, mainly cattle. Many cattle become infected early in life when they are exposed to an environment that is contaminated by other animals shedding the organisms in their feces. Detection of E. coli O157:H7 in feces or contaminated food samples requires tests with high sensitivity, which is increased by the use of monoclonal antibodies. However, the production of concentrated monoclonal antibodies in ascites raises animal welfare concerns, and can be expensive. In this study, single chain of variable fragment (scFv) molecules were developed from hybridoma clones that produce immunoglobulins specific for the LPS and flagella antigen of E. coli O157:H7 using phage display technology. The reactivity of the soluble scFv for their respective antigens was preserved in ELISA and by partial inhibition of bacterial agglutination with polyclonal antiserum. Furthermore, the scFv were able to capture E. coli O157:H7 bacteria demonstrating their potential use in diagnostic assays.     

Shiga toxin of enterohemorrhagic Escherichia coli type O157:H7 promotes intestinal colonization

Enterohemorrhagic Escherichia coli (EHEC) 0157:H7 is a food-borne pathogen that can cause bloody diarrhea and, occasionally, acute renal failure as a consequence of Shiga toxin (Stx) production by the organism. Stxs are potent cytotoxins that are lethal to animals at low doses. Thus, Stxs not only harm the host but, as reported here, also significantly enhance the capacity of EHEC O157:H7 to adhere to epithelial cells and to colonize the intestines of mice. Tissue culture experiments showed that this toxin-mediated increase in bacterial adherence correlated with an Stx-evoked increase in a eukaryotic receptor for the EHEC O157:H7 attachment factor intimin.