LAMP实时浊度法快速检测大肠杆菌方法的建立与优化研究  

Establishment and Optimization of LAMP Real-Time Turbidity Method for Rapid Detection of Escherichia coli

作　　者：陈柱[1] 贺子瑜 何农跃[1] 邓燕[1] 李松[1] CHEN Zhu;HE Ziyu;HE Nongyue;Deng Yan;LI Song(Hunan Key Laboratory of Biomedical Nanomaterials and Devices,Hunan University of Technology,Zhuzhou Hunan 412007,China)

机构地区：[1]湖南工业大学生物医用纳米材料与器件湖南省重点实验室

出　　处：《包装学报》2019年第2期52-58,共7页Packaging Journal

基　　金：湖南省重点研发计划基金资助项目(2017SK2174);国家自然科学基金资助项目(61871180);中国博士后科学基金一等资助项目(2018M630498)

摘　　要：根据大肠杆菌的特异性基因(malB),设计与筛查环介导等温扩增(LAMP)的引物,通过对甜菜碱用量、环引物的浓度、反应温度、dNTPs用量等反应条件的优化,得到较佳的LAMP反应体系,建立了LAMP实时浊度法快速检测大肠杆菌;在较优条件下,先后对9株非大肠杆菌菌株进行特异性检测和5株大肠杆菌的进行同源性检测。此外,结合课题组自行研制的多通道浊度仪,对LAMP扩增产物进行实时浊度监测。结果表明:LAMP实时浊度法检测大肠杆菌的灵敏度达16.010ng/L,与实时荧光定量PCR的检测限相当。因此,建立与优化LAMP实时浊度法可为食品中大肠杆菌的现场快速检测提供了有力手段。According to the specific gene (malB) of Escherichia coli (E.coli), the primers of loop mediated isothermal amplification (LAMP) were designed and screened, and the optimum LAMP reaction system was obtained by optimizing the reaction conditions such as betaine dosage, concentration of loop primer, reaction temperature and dNTPs dosage. Then the optimal method was established for rapid detection of E.coli by LAMP real-time turbidity method. In the optimal conditions, 9 strains of non-Escherichia coli strains were tested for specificity and 5 strains of Escherichia coli were tested for homology. Moreover, using the multichannel turbidity device developed by the research group, the produce of LAMP could be real-time monitored. The results showed that the sensitivity of E.coli was 16.010 ng/L, in line with the detection limit of real-time fluorescence quantitative PCR. Therefore, the optimization and establishment of real-time turbidity LAMP method could provide a powerful approach to rapid field-detection of E.coli in food.

关 键 词：环介导等温扩增 大肠杆菌 实时浊度检测 

分 类 号：TP273[自动化与计算机技术—检测技术与自动化装置]