Development of rapid detection method of viable Escherichia coli and coli-form

活大肠杆菌及大肠菌型快速检测方法的研制

• 批准号: 07660168
• 负责人: MIYAMOTO Takahisa
• 金额: $ 1.73万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660168/
• 关键词: Escherichia coli; coli-form; monoclonal antibody; Rapid detection

Three anti-E.coli monoclonal antibodies were prepared. Among them, MAb-C was most specific to E.coli. The MAb did not react with other bacteria such as Enterobacter aerogenes, Klebsiella pneumoniae, Proteus vulgaris, Serratia marcescens, and Salmonella typhimurium. As a preliminary experiment to develop rapid detection of viable cells, ATP assay was performed on E.coli harvested on a membrane filter. By measuring light emission, about 10^4 CFU of E.coli was detected. The same membrane was treated with peroxidase-labeled MAb-C and light emission was measured after reaction with luminol solution (MAb assay). About 10^3 CFU of E.coli were detected with the Mab assay. When the membrane was incubated in Nutrient broth for 6 hr at 35ﾟC , E.coli was detected at 1 CFU by both methods. Initial cell nember and total light emission counts or total light emission points on the filter was strongly correlated. Correlation coefficients were calculated to be 0.95 and 0.89, respectively, for total light emission counts and total light emission points. In the case of MAb assay, correlation was not so strong between initial cell number and total light emission points (r=0,76). Further work is under way to obtain more specific MAb to E.coli.

三个anti-E。制备大肠杆菌单克隆抗体。其中，单抗- c对大肠杆菌的特异性最强。单抗与其他细菌如产气肠杆菌、肺炎克雷伯菌、普通变形杆菌、粘质沙雷菌和鼠伤寒沙门菌均无反应。作为建立快速检测活细胞的初步实验，对膜过滤器上收获的大肠杆菌进行了ATP测定。通过光发射测量，检测到大肠杆菌约10^4 CFU。用过氧化物酶标记的单克隆抗体- c处理同一膜，与鲁米诺溶液反应后测量发光（单克隆抗体测定）。单抗法检出大肠杆菌约10^3 CFU。当膜在35℃的营养液中孵育6 h时，两种方法在1 CFU时均检测到大肠杆菌。初始细胞数与滤光片上的总光发射点数或总光发射点密切相关。计算出总发光数和总发光点的相关系数分别为0.95和0.89。在单抗试验中，初始细胞数与总发光点之间的相关性不那么强（r=0,76）。目前正在开展进一步的工作，以获得针对大肠杆菌的更特异的单克隆抗体。