**[Multiplex PCR assay]**

Five PCR products of different sizes were amplified simultaneously from five food-borne pathogenic bacteria with the multiplex PCR assay used in this study (Fig. 2). For all of the bacteria tested, the optical density (absorbance value) at 600 nm was 0.010 and 0.080. The different sizes of the amplification products allowed rapid and specific discrimination of *Vibrio parahaemolyticus*, *Salmonella* spp., *Staphylococcus aureus*, *E. coli* O157:H7 and *L. monocytogenes*. The annealing temperature, extension time, and primer concentrations used in this multiplex PCR assay were optimized. The PCR products were separated by agarose gel electrophoresis, and the negative controls used with the multiplex PCR produced negative results. Using the multiplex primers, another single amplification was conducted to confirm the chromosomal DNA from samples contaminated with single specific pathogenic bacteria. In the multiplex PCR with mixed DNA samples, five different bands of specific sizes corresponding to the target genes (Table 2) were detected simultaneously after amplification of the contents of a single tube (Fig. 2).

Fig. 5. Agarose gel electrophoresis showing the result of multiplex PCR amplification of five target gene segments from purified DNA of the five microbial pathogens

M, 100 bp size marker; lane 1, negative control (no template); lane 2, *E. coli* O157:H7 NVRQS; lane 3, *Staphylococcus aureus* KCTC1927; lane 4, *Vibrio parahaemolyticus* KCCM41654; lane 5, *Listeria monocytogenes* ATCC15313; lane 6, *Salmonella enteritidis* ATCC10376; lane 7, Multiplex PCR amplification of all five target genes.
