Novel marker genes for simultaneous detection of Salmonella, EHEC O157:H7, and Cronobacter

Food-borne pathogens represent a significant threat to global food safety and public health. Enterohemorrhagic Escherichia coli (EHEC) O157:H7, Salmonella, and Cronobacter are among the principal bacterial agents responsible for food-borne illnesses. Advances in sequencing technology have made it possible to identify particular genetic markers for these pathogens through large-scale genomic analysis. In this study, two conserved genes, z0340 and sbcC, were screened in silico from extensive bacterial genomes (including 1,460,756 genomes) for EHEC O157:H7 and Salmonella, respectively. Based on these two newly identified markers, combined with previously identified Cronobacter marker gene ygcB, multiplex PCR and multiplex TaqMan quantitative PCR methods were developed and established for the simultaneous detection of EHEC O157:H7, Salmonella, and Cronobacter. The specificity of the multiplex PCR and multiplex TaqMan qPCR was determined using 15 strains of Cronobacter, one strain of EHEC O157:H7, seven strains of Salmonella, and 100 strains of other common environmental bacteria and pathogens. The results showed that two methods demonstrated 100% specificity, both for multiple PCR and TaqMan qPCR. The detection limits of the multiplex PCR and TaqMan qPCR assays were 1 pg/μL and 0.5 pg/μL, respectively. Therefore, the newly developed methods are sensitive and reliable for the simultaneous identification of the three common food-borne pathogens.