**[Specificity of the primer pairs and the multiplex PCR]**

To evaluate the specificity of each oligonucleotide primer pair for its target gene, a PCR assay was carried out by testing all the reference strains reported in Table 2.1. The multiplex PCR was developed specifically and efficiently using amplified reactions and the same PCR program. The reaction was performed in a total volume of 25 l that contained 5 to 15 l (50 ng) of template.

#### **[Food sample processing and multiplex PCR assay]**

A sample of ham (CJ, Seoul, South Korea) from the Korea Food & Drug Administration was used for all tests. Equal concentration of the bacteria were used for inoculation of the ham. *E. coli* O157:H7, *Staphylococcus aureus*, *Listeria monocytogenes*, *V. parahaemolyticus* and *Salmonella typhimurium* were inoculated either single or as two or three species simultaneously. Media bottles (500 ml) containing 25 g of crushed ham were inoculated with bacteria at 100 CFU of each species alone or with 2 X 103 CFU for inoculation of the three species together. inoculated ham was vigorously mixed by shaking for about 30sec to

Studies on PCR-Based Rapid Detection Systems for Salmonella spp. 423

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,

The sensitivity and specificity of the PCR assay were evaluated with 67 food-borne pathogenic bacteria (Table 1). Fig. 3 shows the result of amplification from a representative sample of *Salmonella* spp. The multiplex primer is highly specific for the five pathogenic bacteria target sequence; all *Salmonella* serovars tested produced amplicons of the expected size (678 bp) without spurious priming and without cross-reactivity with non-*Salmonella* species. Results for the other four bacterial species also highly specific (data not shown). Fig. 4 illustrates the detection sensitivities of the multiplex PCR assay, which were evaluated using whole cell cultures of *S. choleraesuis* KCCM41035 and *S. bongori* KCCM41758, cell cultures diluted 10-fold from 1:10 to 1:108 were tested. Based on these results, the multiplex PCR assay detection limits were approximately 105 CFU / ml. Detection results for the other

 **M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23** 

Fig. 6. Specificity for five food pathogenic bacteria using the multiplex primer sets for the

M,100 bp size marker; lane 1, Negative control(no template); lane 2, *S. bongori* KCCM41757; lane 3, *B. subtilis* KCTC2213; lane 4, B. cereus KCTC1526; lane 5, Listeria monocytogenes ATCC15313; lane 6, *L. innocula* ATCC3091; lane 7, *S. enteritidis* ATCC13076; lane 8, *S. typhimurium* KCTC2421; lane 9, *Shigella boydii* ATCC12034; lane 10, *Shigella flexneri* ATCC12022; lane 11, *Shigella flexneri* KCTC2517; lane 12*, Shigella sonnei* KCTC2009; lane 13, *S. enteritidis* KCCM12021; lane 14*, Shigella sonnei* KCTC2518; lane 15, *Shigella sonnei* KCCM41282; lane 16*, S. choleraesuis* KCCM41035; lane 17, *Shigella sonnei* KCCM41282; lane 18, *Y. enterocolitica* KCCM41657; lane 19, *B. cereus* KCTC1661; lane 20, *B. lichniformis* KCTC3006; lane 21, *B. thuringiensis* KCTC1510; lane 22, *Citrobacter fruendii* KCCM11931; lane

The non-autoclaved ham samples were representative of samples that would be collected from a commercial food processing environment. The detection limit for the five pathogens inoculated individually onto non-autoclaved ham was 2 CFU / ml after enrichment. For

Multiplex PCR amplification of all five target genes. **[Specificity and sensitivity for selected primer sets]** 

four bacteria with this assay were similar (data not shown).

detection of *Salmonella* spp.

23, *Listeria murray* ATCC25402.

distribute the bacteria. After inoculation, 225 ml of freshly made LB broth was added to each bottle containing ham. To suspend the bacteria, the bottles were shaken for 10 min at 200 rpm and then incubated at 37°C for 16 hours (Kim *et al*., 2006)*.* Raw pork was also processed as described method above.

The five bacterial species were inoculated simultaneously in raw pork. Water and milk were directly inoculated with five strains; 1 ml of medium containing each strain was added to 9 ml of water and milk and diluted 10 times from 1:10 to 1:108.
