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.