本研究为了研制一种多重PCR检测试剂盒，用于对肠道致病性大肠埃希氏菌（Enteropathogenic Escherichia coli，EPEC）、产志贺毒素大肠埃希氏菌（Shigatoxin-producing E. coli，STEC）和肠道出血性大肠埃希氏菌（Enterohemorrhagic E. coli，EHEC）3种DEC（Diarrheagenic E. coli，DEC）的快速检测。优化所涉及的多重PCR检测体系和构建相应的检测试剂盒，其中的主要检测试剂采用冻干工艺，评价该试剂盒的检测特异性、灵敏度、重复性以及保质期。结果表明，本研究成功建立了针对于3种DEC的检测体系EP和检测体系ST；3种DEC标准菌株和分离菌株均出现明显的目标基因条带，而其他种类DEC标准菌株和分离菌株均未出现目标基因条带；该试剂盒检测管EP和检测管ST的检出限分别可达到1.5×103 cfu/mL和2.1×103 cfu/mL，且二者检测重复率均为100%；该试剂盒可在4 ℃条件下保存12个月，也可在42 ℃环境运输120 h，期间其检测效力不受影响。本研究研制的试剂盒性能好，检测结果稳定、可靠，适用于食品中3种DEC的快速检测。

In order to develop a multiplex PCR detection kit for detecting three types of Diarrheagenic Escherichia coli (DEC), which includes Enteropathogenic E. coli (EPEC), Shigatoxin-producing E. coli (STEC) and Enterohemorrhagic E. coli (EHEC), multiplex PCR reaction system was optimized and their components of the kit were confirmed. The main detection reagents were produced into powder pattern by freeze drying technology, and the specificity, sensitivity, repeatability and shelf life of the kit were evaluated. The results showed that EP and ST reaction systems were successfully established for detection of three types of DEC. All standard strains and isolated strains belonging to the three types of DEC were detected positive and presented obvious specific amplification bands, while other types of DEC were all detected negative. The limit of detection of detection tube EP and ST in the kit were calculated to 1.5×103 cfu/mL and 2.1×103 cfu/mL, respectively, and the testing accordance rates were all 100%. The kit can be stored under 4°C condition for 12 months and transported under 42 ℃ condition for 120 h without efficacy loss. In conclusion, the kit had a good performance with stable and reliable detection results, and it could be applicable for rapid detection of three types of DEC in food samples. Key words:

国家重点研发计划项目(2018YFC1604201)；肇庆市引进西江创新团队项目（肇人才领2018-8）；广东省自然科学基金项目（2017A030310090）