免疫捕获LAMP法快速检测大肠埃希氏O157:H7的研究  

Rapid detection of E.coli O157:H7 by immunocapture and loop-mediated isothermal amplification

作　　者：金婷婷[1] 马福金[1] 袁耀武[1] 

机构地区：[1]河北农业大学食品科技学院,保定071001

出　　处：《食品科技》2013年第8期318-322,共5页Food Science and Technology

基　　金：河北省自然科学基金项目(C2008000216)

摘　　要：目的:建立免疫捕获-LAMP快速检测E.coli O157:H7的方法,并探讨其特异性和灵敏度。方法:制备E.coli O157:H7单克隆抗体,运用酶标板捕获富集待测样品中E.coli O157:H7,以编码该菌鞭毛H7特异性抗原的fliC基因作为靶序列,进行LAMP检测,肉眼观察产物颜色变化并判断检测结果。对3株E.coli O157:H7菌和14株非E.coliO157菌进行特异性检测。结果:只有3株E.coli O157:H7菌显示为阳性,其他菌为阴性。该方法检测E.coli O157:H7的灵敏度为2.5cfu/mL,人工污染全脂牛奶中的E.coli O157:H7检出限为8cfu/mL。结论:这种LAMP前处理方法既大大缩短了增菌时间,又提高了检测结果的准确性和稳定性。Objective： To develop the method for the rapid detection of E.coli O157：H7 by immunocapture and loop-mediated isothermal amplification （LAMP） and to investigate their specificity and sensitivity. Method： The monoclonal antibody specific to E.coli O157 is established after screening and sub- cloning and is enveloped in ELISA plates to capture E.coli O157：H7 in the samples for extracting DNA templates. Basing on the fliC gene encoding the flagellum H7 of E.coli O157：H7 for the target sequences and utilize the detection system which is capable of performing both the amplification and detection （by fluorescence） of LAMP in one platform. Analysis color change to determine test results. Results： The specific detection was carried out in 3 strains of E.coli O157：H7 and 14 strains of non-E.coli O157：H7. It was found that all the 3 strains of E.coli O157：H7 showed positive results, whereas other strains showed negative results. The detecting sensitivity of E. coli O157：H7 was 2.5 cfu/mL. The detection limit of E.coli O157：H7 from artificial contamination milk was 8 cfu/mL. Conclusion： This kind of LAMP pretreatment method not only greatly shorten the time for increasing E.coli O157：H7 and it can improve the detection results of the accuracy and stability.

关 键 词：快速微生物检测 单克隆抗体 免疫捕获LAMP 肠出血性大肠杆菌O157 H7 

分 类 号：TS207.4[轻工技术与工程—食品科学]