To optimize the reaction conditions and to analyze the DNA detection limit of a multiplex PCR method for simultaneous detection of Salmonella, Staphylococcus aureus, Escherichia coli O157∶H7, Listeria monocytogenes and Shigella.MethodsBased on the invA gene of Salmonella, the 16S rDNA gene of Staphylococcus aureus, the eaeA gene of Escherichia coli O157∶H7, the hlyA gene of Listeria monocytogenes and the ipaH gene of Shigella flexneri, five pairs of primers were designed for multiplex PCR amplification. The reaction conditions were optimized and the detection limit was confirmed. ResultsThe optimal concentration of primers for Staphylococcus aureus, Salmonella and Shigella flexneri was 0.25,0.4μmol/L for Listeria monocytogenes, and 0.3μmol/L for Escherichia coli O157∶H7. The optimal magnesium ion concentration was 2.25mmol/L, the annealing temperature was 60℃. The DNA detection limit of this method was 6.4pg for Staphylococcus aureus, 32pg for Salmonella enteritis, 32pg for Escherichia coli O157∶H7, 800pg for Listeria monocytogenes and 160pg for Shigella Flexnei, respectively.ConclusionThrough optimization of reaction conditions and analysis of detection limit, the results provided a basis for the simultaneous detection of these five pathogens and had a significant prospect for application.