**[Oligonucleotides ]**

The oligonucleotide primers were designed by Primer version 3.0 software (Whitehead Institute), referring to Genbank in order to amplify a chromosomal DNA of *Salmonella* spp. (Table 1). The oligonucleotides and all reagents for PCR used in this study were synthesized and purchased from Bioneer (Daejeon, Korea) and Kogene Biotech Inc. (Seoul, Korea).

50 ng of template DNA was used in a 20 l reaction mixture that included 2X SYBR Green I premix Ex Taq (Takara, Japan), 1X ROX Dye (Takara, Japan), 20 pmol of forward and reverse specific primer (Bioneer, Korea). Cycling conditions began with an initial hold at 94°C for 5 min, followed by 35 cycles consisting of 94°C for 30 s, 60°C for 30 s and 72°C for 30 s. and final extension time carry out 72°C for 5 min. Following amplification, melting curves were acquired on the SYBR channel using a ramping rate of 1°C / 30 s for 60 ~ 94°C. The differentiated data were analysed by 7500 software V1.3.0. with the digital filter set as none.

product size; 60, 100, 200, 300, 400, 500, 600 and 700bp Fifth, condition of PCR. To enable simultaneous detection, each PCR products were designed to have different melting

Fig. 9. Strategy for the development of SYBR Green I real-time PCR detection system.

The oligonucleotide primers were designed by Primer version 3.0 software (Whitehead Institute), referring to Genbank in order to amplify a chromosomal DNA of *Salmonella* spp. (Table 1). The oligonucleotides and all reagents for PCR used in this study were synthesized and purchased from Bioneer (Daejeon, Korea) and Kogene Biotech Inc. (Seoul, Korea). 50 ng of template DNA was used in a 20 l reaction mixture that included 2X SYBR Green I premix Ex Taq (Takara, Japan), 1X ROX Dye (Takara, Japan), 20 pmol of forward and reverse specific primer (Bioneer, Korea). Cycling conditions began with an initial hold at 94°C for 5 min, followed by 35 cycles consisting of 94°C for 30 s, 60°C for 30 s and 72°C for 30 s. and final extension time carry out 72°C for 5 min. Following amplification, melting curves were acquired on the SYBR channel using a ramping rate of 1°C / 30 s for 60 ~ 94°C. The differentiated data were analysed by 7500 software V1.3.0. with the digital filter

**3.1 Materials & methods** 

**[Oligonucleotides ]** 

set as none.

temperature, at least 2°C apart from each other (Fig. 1).


Table 3. Oligonucleotide primers for *Salmonella* spp. used in this study
