基于TaqMan探针双重荧光PCR检测食品中鼠伤寒沙门菌和肠炎沙门菌  

Detection of foodborne Salmonella typhimurium and Salmonella enteritidis by diplex real-time PCR using TaqMan probe

作　　者：袁慕云[1] 许龙岩[1] 曹际娟[2] 阳静[1] 张旺[1] 陈碧玲[1] 相大鹏[1] 

机构地区：[1]广东出入境检验检疫局检验检疫技术中心,广州510623 [2]辽宁出入境检验检疫局

出　　处：《卫生研究》2013年第3期491-496,519,共7页Journal of Hygiene Research

摘　　要：目的建立基于TaqMan探针双重荧光PCR检测鼠伤寒沙门菌和(Salmonella typhimurium,ST)和肠炎沙门菌(Salmonella enteritidis,SE)的方法。方法根据ST的STM4599序列(GenBank:AERV01000023.1)和SE特异序列(GenBank:AF370707.1),分别设计引物和探针,ST探针的5'端标记FAM、SE探针的5'端标记VIC,建立基于TaqMan探针双重荧光PCR检测方法。结果 ST和SE的引物和探针分别特异性地扩增出16株ST和15株SE,而28种不同血清型沙门菌和17株变形杆菌等扩增结果均为阴性。ST和SE的双重荧光PCR扩增效率均为94.2%,R2分别为0.998和0.995,最低检测浓度分别达到300 CFU/ml、260 CFU/ml。结论建立的方法特异性好、灵敏度高,整个试验可在31h完成,是快速检测ST和SE的有效方法,可用于食品中ST和SE的特异性检测。Objective To establish a method to detect Salmonella typhimurium（ST） and Salmonella enteritidis（SE） simultaneously with a dual real-time PCR assay using double-color fluorescent TaqMan probes.Methods The primers and probes were designed based on the conservative domain of STM4599 sequence of ST（GenBank： AERV01000023.1） and the specific sequence of SE（GenBank： AF370707.1） respectively.The probes were labeled with reporter dye FAM for ST or VIC for SE at the 5′ end.The dual real-time fluorescence PCR assay was set up and conditions were modified.Results The dual real-time fluorescence PCR method for ST and SE was developed successfully.ST and SE specific primers and probes amplified 16 SE and 15 ST strains,while other 28 different Sa serotypes and 17 negative control Proteus strains showed negative results.The amplification efficiency of ST and SE with the dual fluorescent PCR were all 94.2% and R2 were 0.998 and 0.995 respectively,while the minimum detectable concentration reached 300 CFU/ml for ST and 260 CFU/ml for SE.The entire test can be completed within 31 hours.Conclusion The method is highly specific,sensitive,and fast.The present study thus provides a rapid and effective method to detect ST and SE simultaneously from food samples.

关 键 词：鼠伤寒沙门菌 肠炎沙门菌 双重荧光PCR TAQMAN探针 食品 

分 类 号：R155.5[医药卫生—营养与食品卫生学] TS207.4[医药卫生—公共卫生与预防医学]