生鲜肉中产毒素大肠杆菌多重PCR检测方法构建  

Construction of a Multiplex PCR Method for Detection Enterotoxigenic Escherichia coli in Fresh Meat

作　　者：刘变芳[1] 王涛[1] 王蕊 杜双奎[1] 吕欣[1] 张建新[1] Liu Bianfang;Wang Tao;Wang Rui;Du Shuangkui;La Xin;Zhang Jianxin(College of Food Science and Engineering,Northwest A&F University,Yangling 712100,Shaanxi)

机构地区：[1]西北农林科技大学食品科学与工程学院,陕西杨凌712100

出　　处：《中国食品学报》2018年第12期225-231,共7页Journal of Chinese Institute Of Food Science and Technology

基　　金：杨凌示范区科技计划项目(2017NY-01);陕西省科技攻关项目(K331021302)

摘　　要：大肠杆菌及其致病菌株是生鲜肉中的主要污染菌株,对其检测和控制是保证肉品质量的关键。本研究拟构建基于选择性快速增菌和多重PCR的生鲜肉中产毒素大肠杆菌检测方法。以大肠杆菌种属特异基因uidA、ETEC不耐热肠毒素基因lt和耐热肠毒素基因sta为PCR靶基因,通过引物特异性筛选,ETEC选择性增菌和分离,PCR反应条件优化等手段,对不同肉样中的ETEC进行检测。结果显示:25μL PCR反应体系,退火温度为60℃,引物添加量为0.8μL (10μmol/μL)时,PCR多重扩增稳定性和特异性最佳。经LST肉汤选择增菌和伊红美蓝鉴别平板分离,肉样中ETEC的最低检测限值为4 CFU/10 g。本研究建立的方法操作简便,稳定性、特异性强,可用于生鲜肉中ETEC的快速检测。Escherichia coli and its pathogenic strains are the main pollution strains in fresh meat. The detection and control of these bacteria is the key point to ensure the quality of meat. This study intends to establish a rapid detection method based on selective enrichment and multiplex PCR for Eenterotoxigenic Escherichia coli in fresh meat. The E. coli-specific uid A gene, ETEC heat-labile enterotoxin gene lt and heat-stable enterotoxin gene sta were the three target genes of PCR. Through selection of specific primers, ETEC selective enrichment and separation, and PCR reaction condition optimization study, ETEC was detected from different pork. The results revealed that the stability and specificity of multiplex PCR were the best for the 25 μL reaction system when the annealing temperature was 60 ℃ and the primer addition amount was 0.8 μL(10 μmol/μL). After LST broth enrichment and MAC plate separation, the minimum detection limit of this method is 4 CFU/10 g. This method is simple, stable and specificity, can be used for rapid detection of ETEC in fresh meat.

关 键 词：产毒素大肠杆菌 多重PCR 快速检测 生鲜肉 

分 类 号：TS251.7[轻工技术与工程—农产品加工及贮藏工程]