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建立基于TaqMan探针双重重实时荧光PCR检测肠道沙门氏菌(Salmonella enterica,SP)和肠炎沙门氏菌(Salmonella Enteritidis,SE)的方法。根据SP的aceA基因(Gen Bank:U43344.1)、肠炎沙门氏菌特异序列SEP(GenBank:AF370707.1),分别设计引物和探针,在ace A探针的5′端标记FAM和SEP探针的5′端标记VIC,建立基于TaqMan探针双重荧光PCR检测方法。试验结果,58株29种不同血清型肠道沙门氏菌均扩增出ace A基因扩增曲线,SEP特异性地扩增出15株SE,而28种不同血清型沙门氏菌和17株变形杆菌等阴性对照株扩增结果均为阴性。ace A和SEP的双重荧光PCR扩增效率分别为100%和104%,R2分别为0.999和0.998,最低检测浓度分别达到280 cfu/m L和260 cfu/m L。建立的方法特异性好、灵敏度高,整个试验可在31 h完成,是快速检测SP和SE的有效方法,可用于食品中SP和SE的特异性检测。
To establish a real-time fluorescence double-stranded PCR assay for detection of Salmonella enterica (SP) and Salmonella Enteritidis (SE) based on TaqMan probe. Primers and probes were designed according to the aceA gene of GenBank (GenBank: U43344.1) and the specific sequence of Salmonella enteritidis (SEP) (GenBank: AF370707.1). The probes of FAM and SEP were labeled at the 5 ’ The 5 ’end of the marker VIC, the establishment of TaqMan probe double fluorescence PCR detection method. The test results showed that 58 strains of 29 different serogroup Salmonella enterica were amplified ace A gene amplification curve, SEP-specific amplification of 15 SE, and 28 different serotypes of Salmonella and 17 Proteus negative control Strains amplification results were negative. The double fluorescent PCR amplification efficiencies of ace A and SEP were 100% and 104%, respectively, with R2 of 0.999 and 0.998, respectively, and the minimum detectable concentrations were 280 cfu / m L and 260 cfu / m L, respectively. The established method has good specificity and high sensitivity, and the whole experiment can be completed in 31 h. It is an effective method for rapid detection of SP and SE, and can be used for the specific detection of SP and SE in food.