基于TaqMan探针双重荧光PCR检测食品中肠道沙门氏菌和肠炎沙门氏菌

Development of a Duplex Real-time PCR Method with Taq Man-based Probes for Detecting Salmonella enterica and Salmonella enteritidis

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。

In order to detect Salmonella enterica（SP） and Salmonella enteritidis（SE） using a Taqman-based duplex real-time PCR method, primers and Taqman probes were designed based on the ace A（Gen Bank： U43344.1） sequence of SP and the SEP sequence（Gen Bank： AF370707.1） of SE. Probes were separately labeled with FAM and VIC. The results showed that all 58 Salmonella strains, with 29 different serotypes, could be amplified with the ace A sequence. Only 15 SE strains could be amplified with the SEP primers and probe, while the other 28 strains, with 29 different serotypes of Salmonella, the 17 strains of Proteus, as well as the negative control strains showed negative results. The amplification efficiency of ace A and SEP were 100% and 104%, respectively. R2 values were estimated to be 0.999 and 0.998, respectively. The detection limits of ace A and SEP for this method were 280 cfu/m L and 260 cfu/m L, respectively. The duplex real-time PCR assay developed in this study showed high sensitivity and specificity, and could be used as a rapid and effective method for detecting SP and SE in foods.