畜牧与兽医2018，Vol.50Issue(3)：68-76,9.
采用IMS-RT-PCR方法快速检测肉制品中大肠杆菌O157
Rapid detection of Escherichia coil O157 in meat products by immunomagnetic beads and real-time PCR
摘要
Abstract
In this study,a method was developed for rapid detection of Escherichia coli O157 (EHEC O157) in meat products by immunomagnetic beads separation (IMS) and real-time fluorescence quantitative PCR (RT-PCR).The O-spectific polysaccharides (O-SP) of EHEC O157 were extracted using the hot-phenol water method,and they served as antigen in preparing monoclonal antibodies against EHEC O157 O-SP.The monoclonal antibody (McAb) against EHEC O157 O-SP was coupled to magnetic beads which were activated by 1-ethyl3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS),and the immunomagnetic beads (IMB) were prepared using 150 μg of anti-EHEC O157 O-SP McAb mixed with 1 mg of magnetic beads.The maximal catching of EHEC O157 of 0.2 mg of IMB was 13748.33 CFU.The lowest detectable limit of IMB was 2.2 CFU.Primers and fluorescence probes were designed based on the specific gene of serotype EHEC O157 from rfbE (Genbank login:S83460).The method of IMS combined with RT-PCR was effective in enriching and detecting EHEC O157 in meat products.When the concentration of EHEC O157 in meat products was higher than 1 CFU/g (2.5g meat),IMB could catch it,and RT-PCR could detect EHEC O157 successfully after 3 h culturing in EC broth medium.The whole process took only 6 h to 7 h.The IMS-RT-PCR method has the advantages of shorter duration of detection and higher sensitivity,and it is of great significance for preventing EHEC O157 infection from meat products.关键词
免疫磁珠分离法(IMS)/荧光定量PCR (RT-PCR)/肉制品/肠出血性大肠杆菌O157/O-特异性多糖(O-SP)Key words
immunomagnetic beads separation/real-time fluorescence quantitative PCR/meat product/enterohaemorrhagic Escherichia coli O157/O-spectific polysaccharides分类
农业科技引用本文复制引用
龙梦瑶,蒋蔚,陈永军,熊挺,陈芸,韩阳瑞,凌娇,孙卫东,王权..采用IMS-RT-PCR方法快速检测肉制品中大肠杆菌O157[J].畜牧与兽医,2018,50(3):68-76,9.基金项目
国家重点研发计划(2016YFD0500800-10) （2016YFD0500800-10）
公益性行业(农业)科研专项(20130303045) （农业）
实验动物7种重要质量相关病原体检测新技术的研究(17140900400) （17140900400）
