改良环介导等温扩增技术快速检测肉类中的大肠杆菌O157: H7

Rapid detection of Escherichia Coli O157: H7 in meat using an improved loop-mediated isothermal amplification technology

摘要:

针对大肠杆菌O157: H7 (Escherichia coli O157: H7, E. coli O157: H7) 传统检测方法检测周期长的问题, 建立了肉类中的E. coli O157: H7 的改良环介导等温扩增(LAMP)快速检测方法。以E. coli O157: H7 的O157 特异性抗原rfbE 基因、鞭毛H7 特异性抗原fliC 基因序列作为靶序列,分别设计2 套增加了环引物的改良LAMP 引物序列, 单管同时检测, 通过肉眼观察白色沉淀, 判断检测结果。采用36 株细菌验证了该改良LAMP 引物的特异性。热裂解法提取的DNA 经改良LAMP 体系扩增20 min, 检测E. coli O157: H7 的灵敏度为1.4 CFU/mL, 人工污染肉中的E. coli O157: H7 检出限为1.8 CFU/g。137 份实样中, 检测出1 份E. coli O157: H7 假阳性, 与行业标准SNT0973-2000 符合率达到99.3%。

Abstract:

The detection period of Escherichia coli O157: H7 (E. coli O157: H7) using traditional testing methods is relatively long. Therefore a kind of rapid detection method, namely a loop-mediated isothermal amplification (LAMP) technology with two loop primers detection E. coli O157: H7 in meat, was developed to address this problem. Two sets LAMP primers with loop primers were respectively designed based on the target sequences of rfbE and fliC genes of E. coli O157: H7. The detection results were judged by observing the white precipitate at simultaneous detection of single-tube. The specificity of the improved LAMP was verified using 36 strains of bacteria. DNA extracted by pyrolysis was amplified 20 min by the LAMP system. The detecting sensitivity of E. coli O157: H7 was 1.4 CFU/mL. The detection limit of E. coli O157: H7 from artificial contamination meat was 1.8 CFU/g. One false positive of 137 meat samples was detected, which can obtain compliance rate of 99.3% with the industry standard SNT0973-2000.