**3.2 Results and discussion**

#### **[Primers design and specificity]**

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

Studies on PCR-Based Rapid Detection Systems for Salmonella spp. 429

B. 284 bp

C. 678 bp

Fig. 11. Melting curve analysis for *Salmonella* spp. A ~ C : melting curve of 60, 284, 678 bp

To apply real-time SYBR Green I system to food, various food samples were artificially contaminated by the Salmonella and incubated for enrichment. After overnight enrichment, *Salmonella* spp. gave positive PCR reaction. No signals were observed in negative (un-inoculated) controls. As shown in this result, no other strains but the inoculated *Salmonella enteritidis* was detected (Fig. 4). In conclusion, the SYBR Green I PCR assay combined with DNA extraction using boiling method offers rapid and non-

M, 100 bp ladder; N, negative control; lane 1, hamburger patty contaminated with *Escherichia coli* O157:H7; lane 2, ground poultry with *Escherichia coli* O157:H7; lane 3 , soondae (a sausage made of bean curd and green-bean sprouts stuffed in pig intestine) with *Staphylococcus aureus*; lane 4, kimbob (rice rolled in dried laver) with *Staphylococcus aureus*; lane 5, sea water with *Vibrio parahaemolyticus*; lane 6, shrimp with *Vibrio parahaemolyticus*; lane 7, salad with *Listeria monocytogenes*; lane 8, ice-cream with *Listeria monocytogenes*; lane 9, frozen chicken with *Salmonella enteritidis*.; lane 10, salad with *Salmonella enteritidis*.; lane 11, Soybean paste with *B. cereus*; lane 12, korean red pepper paste with *B. cereus*; lane 13, bottled water with *Yersinia enterocolitica*; lane 14, milk with *Yersinia entercolitica*; lane 15, spring water with *Shigella* spp.; lane 16, oyster with *Shigella* 

**[Application of Real-time SYBR Green** Ⅰ **PCR system to food]** 

sequence-specific detection of amplicons.

spp.; P, Positive control (100 pg)

primer sets

Fig. 10. PCR amplification of *Salmonella* spp*.* using each primer set

M, 100 bp DNA ladder; lane 1, 2, 60 bp primer; lane 3, 4, 137 bp primer; lane 5, 6, 284 bp primer; lane 7, 8, 330 bp primer; lane 9, 10, 424 bp primer; lane 11, 12, 551 bp primer; lane 13, 14, 678 bp primer; lane 15, 16, 787 bp primer; odd lane number, negative control; even lane number, positive control.

#### **[Real-time PCR system]**

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 ABI 7500 PCR machine as a detector and SYBR Green I reagent as a staining dye. First, selection of specific primers; primer size (17~25 mer), hybridization ability, secondary structure within primer, GC contents (40~60%), melting temperature (Tm) (55~65°C). Second, factors affecting Tm; product size, GC content of product. Third, effect of commercial SYBR green reagent; Takara, A&B, Qiagen and in house reagent. Fourth, products size; approximately 60, 100, 200, 300, 400, 500, 600 and 700 bp. Fifth, running conditions of real-time PCR. to enable simultaneous detection, each PCR products were designed to have different melting temperature, at least 2°C apart from each other.

As shown in Fig. 3 primer of 60 bp showed two peaks of positive and negative control at the same position. Primer of 284 bp did not showed non-specific products at all but it was also unavailable because the peak of positive control was too weak. The Tm value was 86.8°C. Only the primer of 678 bp was proved to be available. Although a weak undesired peak was appeared beneath 75°C, it is ignorable because its temperature is sufficiently low. The Tm value was measured as 86.7°C. Therefore, the primer of 678 bp is finally selected for SYBR Green I system.

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

M, 100 bp DNA ladder; lane 1, 2, 60 bp primer; lane 3, 4, 137 bp primer; lane 5, 6, 284 bp primer; lane 7, 8, 330 bp primer; lane 9, 10, 424 bp primer; lane 11, 12, 551 bp primer; lane 13, 14, 678 bp primer; lane 15, 16, 787 bp primer; odd lane number, negative control; even lane

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 ABI 7500 PCR machine as a detector and SYBR Green I reagent as a staining dye. First, selection of specific primers; primer size (17~25 mer), hybridization ability, secondary structure within primer, GC contents (40~60%), melting temperature (Tm) (55~65°C). Second, factors affecting Tm; product size, GC content of product. Third, effect of commercial SYBR green reagent; Takara, A&B, Qiagen and in house reagent. Fourth, products size; approximately 60, 100, 200, 300, 400, 500, 600 and 700 bp. Fifth, running conditions of real-time PCR. to enable simultaneous detection, each PCR products were designed to have different melting temperature, at least 2°C apart from each other. As shown in Fig. 3 primer of 60 bp showed two peaks of positive and negative control at the same position. Primer of 284 bp did not showed non-specific products at all but it was also unavailable because the peak of positive control was too weak. The Tm value was 86.8°C. Only the primer of 678 bp was proved to be available. Although a weak undesired peak was appeared beneath 75°C, it is ignorable because its temperature is sufficiently low. The Tm value was measured as 86.7°C. Therefore, the primer of 678 bp is finally

A. 60 bp

Fig. 10. PCR amplification of *Salmonella* spp*.* using each primer set

number, positive control. **[Real-time PCR system]** 

selected for SYBR Green I system.

Fig. 11. Melting curve analysis for *Salmonella* spp. A ~ C : melting curve of 60, 284, 678 bp primer sets

#### **[Application of Real-time SYBR Green** Ⅰ **PCR system to food]**

To apply real-time SYBR Green I system to food, various food samples were artificially contaminated by the Salmonella and incubated for enrichment. After overnight enrichment, *Salmonella* spp. gave positive PCR reaction. No signals were observed in negative (un-inoculated) controls. As shown in this result, no other strains but the inoculated *Salmonella enteritidis* was detected (Fig. 4). In conclusion, the SYBR Green I PCR assay combined with DNA extraction using boiling method offers rapid and nonsequence-specific detection of amplicons.

M, 100 bp ladder; N, negative control; lane 1, hamburger patty contaminated with *Escherichia coli* O157:H7; lane 2, ground poultry with *Escherichia coli* O157:H7; lane 3 , soondae (a sausage made of bean curd and green-bean sprouts stuffed in pig intestine) with *Staphylococcus aureus*; lane 4, kimbob (rice rolled in dried laver) with *Staphylococcus aureus*; lane 5, sea water with *Vibrio parahaemolyticus*; lane 6, shrimp with *Vibrio parahaemolyticus*; lane 7, salad with *Listeria monocytogenes*; lane 8, ice-cream with *Listeria monocytogenes*; lane 9, frozen chicken with *Salmonella enteritidis*.; lane 10, salad with *Salmonella enteritidis*.; lane 11, Soybean paste with *B. cereus*; lane 12, korean red pepper paste with *B. cereus*; lane 13, bottled water with *Yersinia enterocolitica*; lane 14, milk with *Yersinia entercolitica*; lane 15, spring water with *Shigella* spp.; lane 16, oyster with *Shigella*  spp.; P, Positive control (100 pg)

Studies on PCR-Based Rapid Detection Systems for Salmonella spp. 431

RNase H, two displacement events occur in the presence of four specially designed primers that lead to powerful amplification of target DNA. Since the amplification is initiated only after hybridization of the four primers, the ICA method leads to high specificity for the target sequence. In the CPT method, a DNA-RNA-DNA chimeric probe is hybridized with the target DNA, and the RNA region of the duplex is specifically cleaved by RNase H. The cleaved probe fragments are disassociated from the target DNA and another intact probe is again hybridized and then cleaved. In the cycling events, a single target DNA molecule results in a large number of cleaved probe fragments, which can be designed to generate fluorescent signals (Fig. 1). In the present study, a sensitive and specific iTPA assay for

detecting *Salmonella* spp. in experimentally inoculated food samples was developed.

Fig. 13. The process for the Isothermal target & Probe amplification

The *Salmonella invA* gene (GenBank: EU348369) was used as the target for iTPA primer and probe design. Four primers, two outer and two inner, and one FRET probe which

**[iTPA primers, FRET probe, and reaction conditions]** 

**4.1 Materials & methods** 

Fig. 12. Detection specificity using the *Salmonella* spp. 674 bp primer in contaminated variety food samples

The minimum detection limit was 10 cells / ml with pure culture, which is far more sensitive than conventional PCR which has detection limit of 10,000 cells / ml. In conclusion, we developed a highly sensitive and specific real-time PCR assay for detection of the five food-borne pathogenic bacteria in food samples. This newly developed assay was successfully used to monitor the dynamics of this novel bacterium in food (Abu *et al*, 2005).
