Ecological Studies on Food-Bacteria from Food and Environments

食品和环境中食品细菌的生态学研究

• 批准号: 09460146
• 负责人: UEMURA Takashi
• 金额: $ 5.57万
• 依托单位: Coolage of Agriculture, Osaka Prefecture University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09460146/
• 关键词: Escherichia coli; Clostridium perfringens; enterotoxin; O157; ELISA; flow cytometry; ウェルシュ菌; 病原性大腸菌; カンピロバクター; ウェルシュ菌エンテロトキシン; 0157

(1) To develop a rapid and specific method to detect and/or identify enterohemorrhagic Escberichia coli O 157 : H7, two mouse monoclonal antibodies (MCA) were prepared. Specificities of these two MAbs (1D9 and 3E8) were determined by flow cytometry method (FCM). 3E8 and 1D9 were found to react with E.coli O157 : H7, Citrobacter freundii and Salmonella group N (O : 30), but not with E bermannii With a mixture containing strains of E coil O157 : H7 and E coli O6 : Hl, two different peaks appeared in FCM with MCA, whereas a single peak appeared with polyclonal rabbit antiserum. From these findings, FCM with MCA is suggested to be a rapid, specific, and useful method to detect and identify strain(s) of E celi O157 : H7 in food ingredients (Ref.#1).(2) FCM with fluorescent-labeled anti-CPE antibody was applied to develop a rapid, specific, and convenient method to detect enterotoxin (CPE) exposed on the surface of spores of Clostridium perfringens. The results obtained indicate that FCM can specifically detect CPE exposed on C perfringens spores for a short time. Thus, FCM is found to be a rapid, specific, and convenient assay method for detection of CPE exposed on C perfringens spores, suggesting that it will be hopefully useful to diagnose food poisoning caused by C perfringens (Ref.#2).(3) Sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative estimation of C perfringens enterotoxin (CPE) using* monoclonal* and* polyclonal* antibodies* as* capturing* and* detecting* antibodies* respectively.* The dose- dependent relationship between absorbance at 405 nm and concentration of purified CPE was obtained over the range of 0.5 ng/ml-8.0 ? g/ml. The sandwich ELISA is found to detect crude CPE in culture and CPE in 10% fecal extracts. This method is a convenient, rapid and sensitive for specific detection of GPE(Ref#3).

(1)为建立一种快速、特异的检测和/或鉴定肠出血性大肠杆菌O 157：H7的方法，制备了两株鼠源性单克隆抗体（MCA）。用流式细胞术（FCM）检测1D 9和3E 8单抗的特异性。3E 8和1D 9与大肠杆菌O 157：H7、弗氏柠檬酸杆菌和沙门氏菌群N（O：30）反应，而与伯曼氏大肠杆菌不反应。根据这些发现，建议FCM与MCA是一种快速、特异和有用的方法，用于检测和鉴定食品成分中的大肠杆菌O 157：H7菌株（参考文献# 1）。(2)应用荧光标记抗CPE抗体的流式细胞术建立了一种快速、特异、简便的检测产气荚膜梭菌孢子表面肠毒素（CPE）的方法。结果表明，流式细胞仪可以特异性地检测短时间暴露在产气荚膜梭菌孢子上的CPE。因此，发现FCM是用于检测暴露于产气荚膜梭菌孢子上的CPE的快速、特异性和方便的测定方法，这表明它将有望用于诊断由产气荚膜梭菌引起的食物中毒（参考文献# 2）。(3)以单克隆抗体和多克隆抗体分别作为捕获抗体和检测抗体，建立了双抗体夹心酶联免疫吸附试验（ELISA），用于产气荚膜梭菌肠毒素（CPE）的定量检测。在0.5 ng/ml ~ 8.0 ng/ml范围内，CPE在405 nm处的吸光度与浓度呈剂量依赖关系。G/ml.发现夹心ELISA检测培养物中的粗CPE和10%粪便提取物中的CPE。该方法对于GPE的特异性检测而言是一种方便、快速和灵敏的方法（参考文献3）。

项目成果

KUSUNOKI,Hiromufi, K.KOBAYASHI, T.KITA, T.TAJIMA, S.SUGII, and T.UEMURA: "Analysis of Enterohemorrhagic Escherichia Coli Serotype O157 : H7 by Flow Cytometry Using Monoclonal Antibodies" J.Vet.Med.Sci.60. 1315-1319 (1998)

KUSUNOKI、Hiromufi、K.KOBAYASHI、T.KITA、T.TAJIMA、S.SUGII 和 T.UEMURA：“使用单克隆抗体通过流式细胞术分析肠出血性大肠杆菌 O157 血清型：H7”J.Vet.Med.Sci。

Kusunoki,Hirofumi,K.Kobayashi,T.Kita,T.Tajima,S.Sugii,and T.Uemura: "Analysis of Enterohemorrhagic Escherichia Coli Serotype O157:H7 by Flow Cytometry Using Monoclonal Antibodies." J.Vet.Med.Sci..60. 1315-1319 (1998)

Kusunoki、Hirofumi、K.Kobayashi、T.Kita、T.Tajima、S.Sugii 和 T.Uemura：“使用单克隆抗体通过流式细胞术分析肠出血性大肠杆菌 O157:H7 型。”