**2.1.1 Materials & methods [bacterial strains]**

Bacterial strains were obtained from the American Type Culture Collection (ATCC; Manassas, Va.), the Korean Collection for Type Culture (KCTC; Daejeon, South Korea), and the Korean Culture Center of Microorganisms (KCCM; Seoul, South Korea), Also the strains isolated from various food samples were used in this study (Table 1).

Studies on PCR-Based Rapid Detection Systems for Salmonella spp. 419

Strain Source a Cultural medium

*Bacillus cereus* KCTC1661 LB Broth *Bacillus cereus* KCTC 3624 LB Broth *Salmonella typhimurium* KCTC 2421 LB Broth *Bacillus subtilis* KCTC 3013 LB Broth *Staphylococcus arlettae* KCTC 3588 LB Broth *Citrobacter freundii* KCCM 11931 LB Broth *Bacillus licheniformis* KCTC 3006 LB Broth *Salmonella choleraesuis* KCCM 41575 LB Broth *Shigella sonnei* KCTC 2009 LB Broth *Stphylococcus aureus* KCTC 1916 LB Broth *Salmonella typhimurium* KCTC 2515 LB Broth *Shigella bongori* KCCM 41758 LB Broth *Staphylococcus caprae* KCTC 3583 LB Broth *Salmonella typhimurium* ATCC 14028 LB Broth *Staphylococcus warneri* KCTC 3340 LB Broth *Salmonella enterica* KCTC 2929 LB Broth *Staphylococcus aureus* KCTC 1927 LB Broth *Listeria grayi* ATCC 700545 LB Broth *Listeria ivanovii* ATCC 49953 LB Broth *Listeria grayi* ATCC 25400 LB Broth *Listeria innocuia* ATCC 33091 LB Broth *Listeria murroy* ATCC 25403 LB Broth *Listeria ivanovii* ATCC 49954 LB Broth *Escherichia coli* O157:H7 NVRQ LB Broth *Listeria innocuia* ATCC 33090 LB Broth *Staphylococcus aureus* KCTC 1928 LB Broth

a KCCM, Korean Culture Center of Microorganisms KCTC, Korean Collection for Type Culture ATCC, American Type Culture Collection

 KACC, Korean Agricultural Culture Collection Table 1. Bacterial strains used in this study

tubes and used for DNA extraction (Fig. 1).

NVRQS, National Veterinary Research and Quarantine Service

**[Enrichment procedures for detection of food-borne microorganisms]** 

All food-borne pathogens were grown for 16 hours in LB broth at 37°C in a shaking water bath. Cells were diluted from 1:10 to 1:108 in 10 ml of Luria-Bertani broth and manipulated as described above to make approximate cell count from 10 to 108 CFU / ml. In each dilution ratio, single enrichment broth samples (1 ml) were collected into 1.5 ml micro-centrifuge

All bacterial strains were grown on Luria-Bertani broth (LB; Bactopeptone 10 g, Yeast extract 5 g, and NaCl 10 g, each per Liter) at 37°C. All *Vibrio* species were grown in LB broth with supplementary 2% sodium chloride. Cultures were grown in LB, and a population of visible microorganisms was obtained by plating 10-fold serial dilutions of broth cultures on to plate count agar (Difco, Sparks, USA) and incubating the plates at 37°C for 16 hours. At each sampling dilution ratio, all bacterial cultures were mixed, and 100 l (approximately 107 CFU) of the suspension was used as DNA templates for PCR.


All bacterial strains were grown on Luria-Bertani broth (LB; Bactopeptone 10 g, Yeast extract 5 g, and NaCl 10 g, each per Liter) at 37°C. All *Vibrio* species were grown in LB broth with supplementary 2% sodium chloride. Cultures were grown in LB, and a population of visible microorganisms was obtained by plating 10-fold serial dilutions of broth cultures on to plate count agar (Difco, Sparks, USA) and incubating the plates at 37°C for 16 hours. At each sampling dilution ratio, all bacterial cultures were mixed, and 100 l (approximately 107 CFU) of the suspension was used as DNA templates for PCR.

Strain Source a Cultural medium

*V. algosus* KCCM41677 Trypticase Soy Broth with 2.5% NaCl

*V. diazotrophicus* KCCM41666 Trypticase Soy Broth with 1% NaCl

*V. furnissii* KCCM41679 Trypticase Soy Broth with 1% NaCl

*V. metschnikovii* KCCM41681 Trypticase Soy Broth with 1% NaCl *V. natriegens* KCCM40868 Nutrient Broth with 1.5% NaCl

*V. proteolyticus* KCCM11992 Nutrient Broth with 3% NaCl *V. salmonicida* KCCM41663 Trypticase Soy Broth with 1% NaCl *V. vulnificus* KCCM41665 Trypticase Soy Broth with 1% NaCl

*V. parahaemolyticus* KCCM11965 LB Broth with 1% Nacl *V. parahaemolyticus* KCCM41664 LB Broth with 1% Nacl *V. parahaemolyticus* Inha university LB Broth with 1% Nacl

*Staphylococcus xylosus* KCCM41465 LB Broth *Bacillus licheniformis* KCTC1831 LB Broth *Yersinia enterocolitica* KCCM41657 LB Broth *Staphylococcus aureus* KCCM11764 LB Broth *Staphylococcus cohnii* KCTC3574 LB Broth *Bacillus subtilis* KCTC2213 LB Broth

*V. carchariae* KCCM40865 Marine Broth *V. cholerae* KCCM41626 Nutrient Broth *V. cincinnatiensis* KCCM41683 Marine Broth

*V. fischeri* KCCM41685 Marine Broth *V. fluvialis* KCCM40827 Marine Broth

*V. hollisae* KCCM41680 Marine Broth *V. marinagilis* KCCM41673 Marine Broth *V. marinofulvus* KCCM41674 Marine Broth *V. marinovulgaris* KCCM41675 Marine Broth *V. mediterranei* KCCM40867 Marine Broth

*V. navarrensis* KCCM41682 Marine Broth *V. penaeicida* KCCM40869 Marine Broth

*Vibrio* spp.

Other bacteria


a KCCM, Korean Culture Center of Microorganisms KCTC, Korean Collection for Type Culture ATCC, American Type Culture Collection NVRQS, National Veterinary Research and Quarantine Service KACC, Korean Agricultural Culture Collection

Table 1. Bacterial strains used in this study

#### **[Enrichment procedures for detection of food-borne microorganisms]**

All food-borne pathogens were grown for 16 hours in LB broth at 37°C in a shaking water bath. Cells were diluted from 1:10 to 1:108 in 10 ml of Luria-Bertani broth and manipulated as described above to make approximate cell count from 10 to 108 CFU / ml. In each dilution ratio, single enrichment broth samples (1 ml) were collected into 1.5 ml micro-centrifuge tubes and used for DNA extraction (Fig. 1).

Studies on PCR-Based Rapid Detection Systems for Salmonella spp. 421

The oligonucleotide primers designed with Primer 3.0 software (Whitehead Institute, Cambridge, Mass.) were based on sequences obtained from Genbank and were used to amplify chromosomal DNA for the five food-borne pathogens (Table 2). The oligonucleotides and all reagents for PCRs were synthesized and purchased from Incorporation Bioneer (Daejeon, South Korea) and KoGene BioTech. (Seoul, South Korea).

direction Sequences (5`→3`) Target

CTCATTTGTACTGTTGAAC

AGGCAACCAGTTGTTGAT

GAATCCTCAGTTTTTCAAC

TAGCCGTAACAACCAATAC

AATTTAACAGCTAAAGAGT

TTCATTAAAGAAAAAGTGT

GATAGACTTTTCGACCCAA

TTGCTCAATAATCAGACGA

CTGGCACAAAATTACTTAC

AACTACTGGAGCTGCTTGT

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

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

GCCTAAATAGA

GTTTC

AAATG

TTGGT

ACGAG

CAAAG

AGATG

AACGA

TTTTC

gene

shiga-

PCR product (bp)

*toxR* 219 bp

*invA* 678 bp

*femA* 264 bp

like toxin 208 bp

p60 protein 454 bp

**[Oligonucleotides]** 

*Vibrio* 

*Staphylococcus* 

*aureus*

*Listeria* 

ng) of template.

Strains Primer

*parahaemolyticus* VP Forward

*Salmonella* spp. SAL Forward

*E. coli* O157:H7 EC Forward

*monocytogenes* LM Forward

name

Primer

Reverse

Reverse

Reverse

Reverse

Table 2. Oligonucleotide primers used in this study

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

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

SA Forward Reverse

#### **[Extraction and preparation of DNA templates for PCR assay]**

Individual samples (1 ml) were centrifuged at 10,000 X g for 3 min. The cell pellets were resuspended in RNase free water (100 l) and placed in a 100°C heating block for 20 min. The samples were cooled for 2 min at room temperature and centrifuged at 16,000 X g for 5 min. The supernatant fluids (5 l) were used to make 25 l of a multiplex PCR reaction mixture, which included 5 l of 5 X reaction buffer (2.5 mM MgCl2 and 0.8 mM concentration of each dNTP), 4 l of the primer mixtures of the five food-borne bacteria, 1 l of Super Taq plus polymerase (Rexgene Biotech., Cheongwon, South Korea), and 10 l of DNase free water in a single tube. The multiplex PCR was run for 35 cycles on a Tpersonal cycler (Whatman Biometra, Goettingen, Germany) under the following conditions : denaturation at 94°C for 30 sec, primer annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. The final cycle included an additional 5 min of extension time at 72°C. A 5 l aliquot of the reaction mixture was then electrophoresis on a 2% agarose gel electrophoresis in 0.5 X Tris-borate buffer at 100 V for 25 min. The amplification products were stained with ethidium bromide and visualized by UV trans-illumination.

Fig. 4. Flow diagram of experimental protocols for PCR template preparation
