Development of a Duplex Real-time PCR Method with TaqMan-based Probes for Detecting Salmonella enterica and Salmonella enteritidis
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Abstract:
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 aceA (GenBank: U43344.1) sequence of SP and the SEP sequence (GenBank: 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 aceA 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 aceA and SEP were 100% and 104%, respectively. R2 values were estimated to be 0.999 and 0.998, respectively. The detection limits of aceA and SEP for this method were 280 cfu/mL and 260 cfu/mL, 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.
