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

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,

Studies on PCR-Based Rapid Detection Systems for Salmonella spp. 427

Forward GAA TCC TCA GTT TTT CAA CGT TTC

Before inoculating into food, five bacterial strains were incubated in 5 ml LB broth for overnight. 100 l of each culture broth was inoculated in 25 g of each food material. These food samples were then mixed with 225 ml of LB broth and incubated for overnight. Food segments in sample solution must be removed before assay because PCR can be inhibited by them. Among the 10 samples, water contaminated with *Salmonella* spp. were analyzed without any pre-treatment, other samples were filtered through gauze before assay. 1 ml of each prepared sample solution was transferred to 1.5 ml e-tube and centrifuged at 12,000 rpm for 10 min. The supernatant was removed and the pellet was re-suspended in 500 ml deionized water. Centrifugation and re-suspension in deionized water was performed one more time for exact assay. 150 l of Deionized water and 50 l of 10% chelex resin was added to the pellet and mixed throughly. The solution was heated at 100°C for 10 min, and centrifuged at 12,000 rpm for 10 min. 5 l aliquot of this solution was taken for SYBR Green I assay. PCR using ABI 7500 (Perkin-elmer, USA) was cycled 35 times as follows: 30 sec denaturation at 94°C, 30 seconds annealing at 60°C, and 30 seconds polymerization at 72°C. The products of real-time PCR were run on 2% agarose gel electrophoresis and melting curves were acquired on the SYBR channel using a

Fig. 2 is the result of PCR reaction performed with various primer sets which are designed for *Salmonella* spp. Specific PCR reaction was observed with primer sets of which product sizes are 60 bp, 284 bp and 678 bp, respectively. However, primer sets of 137 bp, 330 bp and 551 bp showed non-specific products in the place of negative control, meaning that these primers are not available. As for the primers of 424 bp, bacterial DNA was not amplified.

CCA GAC GAA AGA GCG TGG TAA GAA GCC CGA ACG TGG CGA

GTA TGC CCG GTA AAC AGA TGA GT AAA GGA ACC GTA AAG CTG GCT GGG TCA TCC CCA CCG AAA TAC

(5' → 3') Primer sequence Size

(bp)

Strains Direction

Reverse

**[SYBR Green I PCR assay using ABI 7500] [Detection studies with diversity food samples]** 

ramping rate of 1°C / 30 seconds for 60 ~ 94°C.

Therefore, primer sets of 60 bp, 284 bp, and 678 bp were selected.

**3.2 Results and discussion [Primers design and specificity]** 

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

*Salmonella* spp.

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

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