**[Specificity and sensitivity for selected primer sets]**

422 Salmonella – A Dangerous Foodborne Pathogen

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

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

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

 **M 1 2 3 4 5 6 7** 

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

ml of water and milk and diluted 10 times from 1:10 to 1:108.

amplification of the contents of a single tube (Fig. 2).

as described method above.

**2.1.2 Results and discussion** 

**[Multiplex PCR assay]** 

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

 **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 detection of *Salmonella* spp.

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 23, *Listeria murray* ATCC25402.

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

Studies on PCR-Based Rapid Detection Systems for Salmonella spp. 425

This multiplex PCR assay offers the advantages of significantly short processing time and saving cost. Only one composite DNA sample is required rather than separate samples for each target gene to be analyzed (Kim *et al*., 2006)*.* To test the efficacy of this PCR assay for detecting pathogenic bacteria in food, *Salmonella typhimurium* ATCC19585 (10 CFU / g of food) was inoculated into samples of selected foods (milk, raw pork and raw chicken) that had been previously screened for detectable pathogenic microbial contamination. The inoculated samples were then incubated aerobically at 37°C for 8 hours (enrichment culture step). The PCR assay detected bacterial cells in all inoculated samples. However, when a 5 hours instead of 8 hours enrichment culture step was used, no bacteria were

Thus, our PCR assay requires at least 8 hours of enrichment to detect the added pathogenic bacteria in these foods with a detection sensitivity ranging from 10 to 100 CFU / g. Therefore, the enrichment step is required in this PCR protocol for detection of food-borne pathogenic bacteria. The five specific primer sets tested for *Vibrio parahaemolyticus*, *Salmonella* spp., *Staphylococcus aureus*, *E. coli* O157:H7 and *L. monocytogenes* can be used specifically and simultaneously. These five food pathogens were clearly detected from both culture medium artificially inoculated water, milk and raw pork. Thus, the protocol developed in this study could have important application for the rapid and simultaneous detection and identification of up to five food-borne pathogenic bacteria in many foods. This simple method is expected to enable rapid risk assessment of pathogen contamination of foods at a low cost. The cost of detection could be reduced from the \$ 50 (approximately \$ 10 per pathogen) for the traditional method to less than \$ 1 for this multiplex PCR method.

**3. The development of rapid real-time PCR detection system for** *Salmonella* 

Previously, we developed multiplex conventional PCR assay from the conventional PCR methods (Kim *et al*., 2007). Conventional PCR methods for the detection of food-borne bacterial pathogens are time consuming and insensitive that it can not provide adequate screening of samples for the presence of potential pathogens. With the advent of automated real-time PCR suspected food-borne contaminants can be detected in less than an hour. This technique, using TaqMan PCR, has been successfully adapted for the detection of pathogenic bacteria, including *Salmonella enterica*, *Listeria monocytogenes*, *Escherichia coli*  O157:H7, and *Yersinia pestis* (Bassler *et al*, 1995; Bellin *et al*, 2001; Higgins *et al*, 1998; Hoorfar *et al*, 2000; Jothikumar *et al*, 2002; Knutsson *et al*, 2002; Oberst *et al*, 1998; Sharma *et al*, 1999). Can there be a better method which has the same sensitivity with nested PCR and can be performed with one PCR reaction? It will be more effective if there is more sensitive optical instrument and staining dye which can detect very small amount of product than naked eyes and EtBr. Micro PCR, which was developed for this purpose, uses real-time PCR machine as a detector and SYBR Green reagent as a staining dye. Real-Time PCR is currently used for the diagnosis of *Escherichia coli* strain O157:H7 (Ibekwe *et a*l, 2002) and *Plesimonas shigelloides* (Loh *et al*, 2001) in stool specimens. To develop micro PCR, following factors were studied. First, selection of specific primers; primer size (17~25 mer), hybridization ability, secondary structure within primer, GC content (40~60%), melting temperature (Tm) (55~65°C). Second, factors affecting Tm; product size, GC contents of product. Third, effect of commercial SYBR Green reagent; Takara, A&B, Qiagen and in house reagent. Forth,

detected (data not shown).

**spp.** 

non-autoclaved ham incubated with two or three organisms together, the sensitivity was the same as that achieved when the pathogens were evaluated singly (Fig. 5). Without enrichment of the bacterial culture, the detection limits after inoculation of non-autoclaved ham with *E. coli* O157:H7, *S. aureus* and *L. monocytogenes* together were 20,000 cells, respectively (data not shown).

Fig. 7. Sensitivity of the multiplex PCR assay for *Salmonella choleraesuis* KCCM41035 (A) and *Salmonella bongori* KCCM41757 (B)

M, 100 bp size marker; lane 1, 1.2 X 108 CFU / ml; lane 2, 1.2 X 107 CFU / ml; lane 3, 1.2 X 106 CFU / ml; lane 4, 1.2 X 105 CFU / ml; lane 5, 1.2 X 104 CFU / ml; lane 6, 1.2 X 103 CFU / ml; lane 7, 1.2 X 102 CFU / ml; lane 8, 1.2 X 10 CFU / ml; lane 9, 1.2 X 108 CFU / ml; lane 10, 1.2 X 107 CFU / ml; lane 11, 1.2 X 106 CFU / ml; lane 12, 1.2 X 105 CFU / ml; lane 13, 1.2 X 104 CFU / ml; lane 14, 1.2 X 103 CFU / ml; lane 15, 1.2 X 102 CFU / ml; lane 16, 1.2 X 10 CFU / ml

**[Validity of the multiplex PCR assay for food samples]** 

Fig. 8. Amplification products obtained with the multiplex PCR assay

M, 100 bp size marker; N, negative control; lane 1, PCR with *E. coli* O157:H7; lane 2, PCR with *E. coli* O157:H7 DNA (100 pg); lane 3, PCR with *S. aureus*; lane 4, PCR with *S. aureus*  DNA (100 pg); lane 5, PCR with *L. monocytogenes*; lane 6, PCR with *L. monocytogenes* DNA (100 pg); lane 7, PCR with 100 pg DNA each from *E. coli* O157:H7, *S. aureus* and *L. monocytogenes*; lane 8, PCR with 100 pg DNA each from *E. coli* O157:H7, *L. monocytogenes* and *Salmonella typhimurium*; lane 9, PCR with 100 pg DNA each from *S. aureus* and *V. parahaemolyticus*

non-autoclaved ham incubated with two or three organisms together, the sensitivity was the same as that achieved when the pathogens were evaluated singly (Fig. 5). Without enrichment of the bacterial culture, the detection limits after inoculation of non-autoclaved ham with *E. coli* O157:H7, *S. aureus* and *L. monocytogenes* together were 20,000 cells,

M 1 2 3 4 5 6 7 8 M 9 10 11 12 13 14 15 16

(A) (B)

Fig. 7. Sensitivity of the multiplex PCR assay for *Salmonella choleraesuis* KCCM41035 (A) and

M, 100 bp size marker; lane 1, 1.2 X 108 CFU / ml; lane 2, 1.2 X 107 CFU / ml; lane 3, 1.2 X 106 CFU / ml; lane 4, 1.2 X 105 CFU / ml; lane 5, 1.2 X 104 CFU / ml; lane 6, 1.2 X 103 CFU / ml; lane 7, 1.2 X 102 CFU / ml; lane 8, 1.2 X 10 CFU / ml; lane 9, 1.2 X 108 CFU / ml; lane 10, 1.2 X 107 CFU / ml; lane 11, 1.2 X 106 CFU / ml; lane 12, 1.2 X 105 CFU / ml; lane 13, 1.2 X 104 CFU /

ml; lane 14, 1.2 X 103 CFU / ml; lane 15, 1.2 X 102 CFU / ml; lane 16, 1.2 X 10 CFU / ml

M N 1 2 M N 3 4 M N 5 6 M N 7 8 9

M, 100 bp size marker; N, negative control; lane 1, PCR with *E. coli* O157:H7; lane 2, PCR with *E. coli* O157:H7 DNA (100 pg); lane 3, PCR with *S. aureus*; lane 4, PCR with *S. aureus*  DNA (100 pg); lane 5, PCR with *L. monocytogenes*; lane 6, PCR with *L. monocytogenes* DNA (100 pg); lane 7, PCR with 100 pg DNA each from *E. coli* O157:H7, *S. aureus* and *L. monocytogenes*; lane 8, PCR with 100 pg DNA each from *E. coli* O157:H7, *L. monocytogenes* and *Salmonella typhimurium*; lane 9, PCR with 100 pg DNA each from *S. aureus* and *V.* 

Fig. 8. Amplification products obtained with the multiplex PCR assay

**[Validity of the multiplex PCR assay for food samples]** 

respectively (data not shown).

*Salmonella bongori* KCCM41757 (B)

*parahaemolyticus*

This multiplex PCR assay offers the advantages of significantly short processing time and saving cost. Only one composite DNA sample is required rather than separate samples for each target gene to be analyzed (Kim *et al*., 2006)*.* To test the efficacy of this PCR assay for detecting pathogenic bacteria in food, *Salmonella typhimurium* ATCC19585 (10 CFU / g of food) was inoculated into samples of selected foods (milk, raw pork and raw chicken) that had been previously screened for detectable pathogenic microbial contamination. The inoculated samples were then incubated aerobically at 37°C for 8 hours (enrichment culture step). The PCR assay detected bacterial cells in all inoculated samples. However, when a 5 hours instead of 8 hours enrichment culture step was used, no bacteria were detected (data not shown).

Thus, our PCR assay requires at least 8 hours of enrichment to detect the added pathogenic bacteria in these foods with a detection sensitivity ranging from 10 to 100 CFU / g. Therefore, the enrichment step is required in this PCR protocol for detection of food-borne pathogenic bacteria. The five specific primer sets tested for *Vibrio parahaemolyticus*, *Salmonella* spp., *Staphylococcus aureus*, *E. coli* O157:H7 and *L. monocytogenes* can be used specifically and simultaneously. These five food pathogens were clearly detected from both culture medium artificially inoculated water, milk and raw pork. Thus, the protocol developed in this study could have important application for the rapid and simultaneous detection and identification of up to five food-borne pathogenic bacteria in many foods. This simple method is expected to enable rapid risk assessment of pathogen contamination of foods at a low cost. The cost of detection could be reduced from the \$ 50 (approximately \$ 10 per pathogen) for the traditional method to less than \$ 1 for this multiplex PCR method.
