乳粉中阪崎肠杆菌污染检测试剂盒的研制  

Development of a Test Kit for Rapid Detection of Enterobacter sakazakii Contamination in Milk Powder

作　　者：宋春美[1] 朱政辉[1] 李建武[1] 刘箐[1] 

机构地区：[1]上海理工大学医疗器械与食品学院,上海200093

出　　处：《食品科学》2016年第24期233-238,共6页Food Science

基　　金：上海市"科技创新行动计划"长三角科技联合攻关领域项目;中国博士后基金项目(2015M581637);2016年度上海理工大学国家级项目培育基金项目(16HJPY-QN09)

摘　　要：目的：以乳粉中污染的阪崎肠杆菌（Enterobacter sakazakii）为检测目标,开发一种能快速检测其污染的试剂盒。方法：以两株阪崎肠杆菌单克隆抗体分别为包被抗体和检测抗体,建立双抗体夹心酶联免疫吸附实验方法,优化各个反应条件,制成在2 h内能发生显色反应的检测试剂盒。结果：包被抗体的最佳工作质量浓度为10？μg/mL,检测抗体的最佳稀释倍数为1∶5 000。试剂盒的检测限为10^4 CFU/mL,在10^4～10^8 CFU/mL范围内OD450 nm值与阪崎肠杆菌浓度的常用对数值呈线性关系。特异性实验中,试剂盒可检出3株阪崎肠杆菌标准菌株,对其他9株食品中常见致病菌的检测均呈阴性。模拟带菌实验中,初始菌浓度为1 CFU/mL的模拟样品经过8 h前增菌后,通过该试剂盒便可检出。该试剂盒的批内、批间变异系数均小于6%,至少可在室温保存6个月。结论：该双抗体夹心试剂盒具有灵敏度高、特异性好、精密度高、稳定性强等优点,可应用于乳粉中阪崎肠杆菌的快速检测。Purpose： To develop a rapid detection kit for Enterobacter sakazakii contamination in milk powder.Methods： A double-antibody sandwich ELISA assay was developed using two monoclonal antibodies against E.sakazakii as the capture antibody and detection antibody,respectively.A test kit that could show a color reaction within 2 h was developed through the optimization of the ELISA reaction.Results： The optimum working concentration of the capture antibody was 10 μg/mL and the optimum dilution of the detection antibody was 1：5 000.The limit of detection（LOD） of the kit was 10^4 CFU/mL and a linear relationship between OD450nm values and the lg values of E.sakazakii concentration was observed in the range of 10^4–10^8 CFU/mL.In specificity test,three reference strains of E.sakazakii could be detected by the kit whereas other nine strains of common foodborne pathogens were detected as negative.The LOD of the kit was substantially improved to 1 CFU/mL of the original bacterial content after pre-incubation of model samples in broth for 8 h.Both the intra-assay and inter-assay coefficients of variation of the kit were lower than 6%.It could be stored at room temperature for at least six months.Conclusions： The established double-antibody sandwich ELISA kit is suitable for the rapid detection of E.sakazakii in milk powder with high sensitivity,high specificity,high precision and strong stability.

关 键 词：双抗体夹心法 酶联免疫吸附试剂盒 灵敏 快速检测 致病菌 

分 类 号：TS201.3[轻工技术与工程—食品科学]