**1.2.3 Isothermal PCR**

Recently, Jung *et al*. (2010) developed a new highly sensitive and specific isothermal amplification and detection system called isothermal target and probe amplification (iTPA) by employing DNA-RNA-DNA chimeric primers and a FRET (fluorescence Resonance Energy Transfer) probe. The iTPA method is based on a combination of novel isothermal chain amplification (ICA) and FRET cycling probe technology (CPT) (Fig. 3).

Studies on PCR-Based Rapid Detection Systems for Salmonella spp. 417

**2.1 Rapid and simultaneous detection of five pathogenic bacteria by a novel multiplex PCR assay:** *Salmonella* **spp.,** *Escherichia coli* **O157:H7,** *Listeria monocytogenes***,** 

According to Centers for Disease Control (CDC), about 5 millions food mediated diseases are killing 4,000 people every year. *Salmonella* was the most frequently found pathogenic bacteria in food poisoning : 1 ~ 4 millions of people were infected, 2,000 (0.1%) of them were dead. *Salmonella* is an important pathogen associated with bacterial foodborne outbreaks in the United states, accounting for 24% of all food outbreaks and 18% of produce-related outbreaks between 1990 and 2009 (Center for Science in the Public Interest, 2009). An outbreak in 2009 associated with *Salmonella*-contaminated peanut butter and peanut containing products caused nine deaths in 46 states as of 17 March 2009. This outbreak led to the largest recall of food items in the United States resulting in over 2100 products being voluntarily recalled by more than 200 companies (FDA, 2009). Recently, more than 500 million eggs were recalled after dangerous levels of *Salmonella* were detected in the eggs from two Iowa producers, who distributed the eggs in 14 US states. Nearly 2000 illnesses were reported between May and July 2010 (CDC, 2010). Food poisoning by E. coli O157:H7 broke out in 10000 people, 300 of them were dead. As for Listeria monocytogenes, 1500 people were infected and 400 were dead. This shows that stock farm products which were contaminated by these four bacteria (*E. coli* O157:H7, *Salmonella* spp., *Listeria monocytogene* and *Staphylococcus aureus*) is seriously threatening consumer's health. In korea, 50% of food poisoning are caused by meat or processed meat products, and *Salmonella* strains (50%), *S. aureus* (20%) are two major sources. Different molecular targets have been used to characterize the presence of food-borne pathogenic bacteria. In this study, genes encoding the virulence determinants and their expression regulator have been used to characterize numerous bacteria. A molecular test based on the detection of shiga-like toxin (verotoxin type II), femA (cytoplasmic protein), toxR (trans-membrane DNA binding protein), iap (invasive associative protein), and invA (invasion protein A) genes has been applied for identification of *E. coli* O157:H7 (Jinneman *et al*., 2003; Kaneko *et al*., 2001; Karpman *et al*., 1998; Schmidt *et al*., 1995; Wang *et al*., 2002), *Staphylococcus aureus* (Mehrotra *et al*., 2000), *Vibrio parahaemolyticus* (Karpman *et al*., 1998; Cabrera-Garcia *et al*., 2004), *Listeria monocytogenes* (Bubert *et al*., 1992; Bubert *et al*., 1999; Volokhov *et al*., 2002), and *Salmonella* 

To our knowledge, there is not a single acceptable method which is available to detect these five food-borne pathogenic bacteria simultaneously in food samples. The objective of the present work, therefore, was to establish a multiplex PCR assay method to detect the specific bacterial genus simultaneously and to analyze their distribution in contaminated foods. Our results indicate, that this method is rapid and specific for the simultaneous detection of *E. coli* O157:H7, *Staphylococcus aureus*, *Vibrio parahaemolyticus*, *Listeria* 

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

**2. Various PCR methods approaches for the detection of** *Salmonella* **spp.** 

*Staphylococcus aureus* **and** *Vibrio parahaemolyticus*

*spp*. (Chiu *et al*., 1996).

*monocytogenes* and *Salmonella* spp.

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

isolated from various food samples were used in this study (Table 1).

Fig. 3. Scheme of the Isothermal Target and Probe Amplification (iTPA) system

In the ICA method, which relies on the strand displacement activity of DNA polymerase and the RNA-degrading activity of RNase H, two displacement events occur in the presence of four specially designed primers that lead 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 fragment is disassociated from the target DNA and another intact probe is again hybridized and then cleaved. In this cycling event, a single target DNA molecule results in a large number of cleaved probe fragments, which can be designed to generate fluorescence signals (Kim *et al*. 2011).

Fig. 3. Scheme of the Isothermal Target and Probe Amplification (iTPA) system

fluorescence signals (Kim *et al*. 2011).

In the ICA method, which relies on the strand displacement activity of DNA polymerase and the RNA-degrading activity of RNase H, two displacement events occur in the presence of four specially designed primers that lead 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 fragment is disassociated from the target DNA and another intact probe is again hybridized and then cleaved. In this cycling event, a single target DNA molecule results in a large number of cleaved probe fragments, which can be designed to generate
