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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 for the detection of Salmonella typhimurium (ST) and Salmonella enteritidis (SE) by TaqMan probe double fluorescent PCR. Methods Primers and probes were designed according to ST4545 (GenBank: AERV01000023.1) and SE specific sequence (GenBank: AF370707.1). The 5 ’end of ST probe was labeled with FAM and the 5’ end of SE probe VIC, a double fluorescent PCR detection method based on TaqMan probe was established. Results The primers and probes of ST and SE respectively amplified 16 strains of ST and 15 strains of SE, while the results of 28 strains of Salmonella serotype and 17 strains of Proteus were negative. The double fluorescent PCR amplification efficiencies of ST and SE were 94.2%, R2 were 0.998 and 0.995 respectively, and the lowest detection concentrations were 300 CFU / ml and 260 CFU / ml, respectively. Conclusion The established method has good specificity and high sensitivity. The whole experiment can be completed in 31h. It is an effective method for rapid detection of ST and SE, and can be used for the specific detection of ST and SE in food.