摘要
目的建立高效、特异、简便、快速的四重实时荧光PCR（real—timePCR）检测体系同时检测沙门菌、金黄色葡萄球菌、单核细胞增生李斯特菌、大肠杆菌0157：H74种常见的主要食源性致病菌。方法从GenBank数据库中下载沙门菌侵袭性基因ssaR、金黄色葡萄球菌肠毒素nuc基因、单增李斯特菌的hlyA基因、大肠杆菌0157：H7的rfbE基因序列，设计引物和探针，利用HAND系统和多色探针编码技术，通过对PCR反应的组分和条件进行优化，建立针对肉类食品中4种食源性致病菌四重实时荧光PCR体系。结果成功建立了检测肉类食品中4种食源性致病菌四重实时荧光体系，DNA和细菌纯培养的最低检测限达到1～100fg／PCR和1～6．25CF-U／PCR，特异性均达98％以上。结论本文利用HAND系统和多色探针编码技术相结合，构建了针对4种食源性致病菌四重实时荧光PCR体系。可用于食品中食源性致病菌的快速筛查和食源性疾病暴发的快速诊断。
Objective To develop an efficient, specific, simple and fast multiplex real- time PCR assay for the detection of 4 common foodborne pathogens： Salmonella, Staphyloccocus aureus , Listeria rnonocytogenes and E. coli O157 ：H7. Methods A set of primers and probes were designed according to the sequences of Salmonella ssaR, Staphyloccocus aureus nuc, Liste- ria rnonocytogenes hlyA, E. coli O157：H7 rfbE genes in Genebank, and the four- color real- time PCR system was established with HAND system and Multicolor Combinational Probe Coding （MCPC） technology. Then, the components and condition of the PCR assay were optimized. Results A multiplex real- time PCR assay was developed for the detection of 4 foodborne pathogens in meat food. The lowest detection limit of DNA and pure cultures of bacteria reached 1 - 100 fg/PCR and 1 - 6.25 CFU/PCR respectively, with the detection specificity higher than 98%. Conclusions A multiplex real - time PCR assay based on HAND system and MCPC technology is developed to simultaneously identify 4 foodbome pathogens, which can be ap- plied to the rapid detection of fcxxtborne pathogens in food and rapid diagnosis of foodborne disease outbreak
出处
《实用预防医学》
CAS
2015年第8期905-908,共4页
Practical Preventive Medicine
基金
国家自然科学基金(81071433)
