**[Oligonucleotides]**

420 Salmonella – A Dangerous Foodborne Pathogen

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

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

ethidium bromide and visualized by UV trans-illumination.

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

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).


Table 2. Oligonucleotide primers used in this study
