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

制备了三株抗大肠杆菌单克隆抗体。其中，MAb-C对大肠杆菌的特异性最强。单克隆抗体不与其他细菌如产气肠杆菌、肺炎克雷伯菌、普通变形杆菌、粘质沙雷氏菌和鼠伤寒沙门氏菌反应。作为开发活细胞快速检测的初步实验，对在膜滤器上收获的大肠杆菌进行ATP测定。通过测量光发射，检测到约10^4 CFU的大肠杆菌。用过氧化物酶标记的MAb-C处理相同的膜，并在与鲁米诺溶液反应后测量光发射（MAb测定）。Mab检测试剂盒检出约10^3 CFU大肠埃希菌。当膜在营养肉汤中于35 ℃下孵育6小时时，两种方法均检出1 CFU的大肠杆菌。初始细胞计数和滤光片上的总发光计数或总发光点强烈相关。对于总发光计数和总发光点，计算出的相关系数分别为0.95和0.89。在单克隆抗体测定的情况下，初始细胞数和总发光点之间的相关性不是那么强（r= 0.76）。目前正在进行进一步的工作，以获得针对大肠杆菌的更特异性单克隆抗体。

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.