五种食源性致病菌多重PCR的条件优化和检出限分析  

Optimization of multiplex PCR method for five food-borne pathogens and analysis of its detection limit

作　　者：陈娟[1] 唐俊妮[1] 李键[1] 陈丽君[1] 

机构地区：[1]西南民族大学生命科学与技术学院,四川成都610041

出　　处：《中国食品卫生杂志》2014年第2期137-141,共5页Chinese Journal of Food Hygiene

基　　金：教育部新世纪人才支持计划(NCET-11-0847);国家自然科学基金(31371781);国家科技重大专项(201203009)

摘　　要：目的对沙门菌、金黄色葡萄球菌、大肠杆菌O157∶H7、单核增生李斯特菌和福氏志贺菌5种食源性致病菌的多重PCR反应条件进行优化并分析方法的DNA检测限。方法根据沙门菌的invA基因、金黄色葡萄球菌的16S rDNA基因、大肠杆菌O157∶H7的eaeA基因、单核增生李斯特菌的hlyA基因和福氏志贺菌的ipaH基因设计5对特异性引物进行多重PCR,对反应条件包括镁离子浓度、引物浓度和退火温度进行优化试验,并确定该检测方法的检出限。结果最佳反应条件为金黄色葡萄球菌、沙门菌和福氏志贺菌引物浓度为0.25μmol/L,单核增生李斯特菌引物浓度为0.4μmol/L,大肠杆菌O157∶H7引物浓度为0.3μmol/L,Mg2+浓度为2.25 mmol/L,退火温度为60℃;在此条件下金黄色葡萄球菌、肠炎沙门菌、大肠杆菌O157∶H7、单核增生李斯特菌、福氏志贺菌的多重PCR的DNA检出限分别为6.4、32、32、800和160 pg。结论通过对5种引物的PCR条件进行优化和检出限的分析,为食品中该5种致病菌的快速检测奠定基础,对实际应用具有指导意义。Objective To optimize the reaction conditions and to analyze the DNA detection limit of a multiplex PCR method for simultaneous detection of Salmonella, Staphylococcus aureus, Escherichia coli O157 ： HT, Listeria monocytogenes and Shigella. Methods Based on the invA gene of Salmonella, the 16S rDNA gene of Staphylococcus aureus, the eaeA gene of Escherichia coli O157 ： H7, the hlyA gene of Listeria monocytogenes and the ipaH gene of Shigellaflexneri, five pairs of primers were designed for multiplex PCR amplification. The reaction conditions were optimized and the detection limit was confirmed. Results The optimal concentration of primers for Staphylococcus aureus, Salmonella and Shigella flexneri was O. 25,0.4 pLmol/L for Listeria monocytogenes, and O. 3 ~moL/L for Escherichia coli O157 ： H7. The optimal magnesium ion concentration was 2. 25 retool/L, the annealing temperature was 60 ~C. The DNA detection limit of this method was 6.4 pg for Staphylococcus aureus, 32 pg for Salmonella enteritis, 32 pg for Escherichia coli O157：H7, 800 pg for Listeria monocytogenes and 160 pg for Shigella Flexnei, respectively. Conclusion Through optimization of reaction conditions and analysis of detection limit, the results provided a basis for the simultaneous detection of these five pathogens and had a significant prospect for application.

关 键 词：多重PCR 扩增条件 优化 检出限 食源性致病菌 食品安全 

分 类 号：R346[医药卫生—基础医学]