**4.5 Nucleic acid hybridization**

382 Salmonella – A Dangerous Foodborne Pathogen

Knabel 2007). Lastly, and perhaps the largest impact that mPCR may provide in a near future is in the rapid and simultaneous detection of *Salmonella* concurrently with other bacterial pathogens. For instance, Gilbert et al. (2003) established a mPCR assay in order to detect *Salmonella* along with *Campylobacter jejuni*, and *E. coli* O157:H7 in a variety of raw and ready-to-eat food products. The primers amplified a single product from each target bacterium. More recently, Kim et al. (2007) developed a novel mPCR assay for the simultaneous screening of five foodborne pathogenic bacteria including *Salmonella*. Specific primers for mPCR amplification of the Shiga-like toxin gene (*Stx2*), *femA* (cytoplasmic protein), *toxR* (transmembrane DNA binding protein), *iap* (invasive associative protein), and *invA* genes were designed to allow simultaneous detection of *E. coli* O157:H7, *S. aureus*, *Vibrio parahaemolyticus*, *L. monocytogenes*, and *Salmonella* spp., respectively. Furthermore, the detection of all five food borne pathogenic bacteria could be completed in less than 24 h. Similar approaches have been described by others utilizing various primer sets for a variety

Thus far, there is no correlation between viability and detection as provided by PCR assays. The amplification of genomic DNA by PCR has been shown to be inappropriate for distinguishing viable from non-viable bacteria owing to DNA stability over time (Masters et al. 1994). Furthermore, the detection of pathogens by PCR in food samples often requires additional evidence of viability before risk can be assigned. In an effort to address the issue of viability, many researchers turned to RNA amplification methods using mRNA as a target since it is a molecule with a very short half-life of 0.5 to 2 minutes due to the rapid degradation by endogenous RNases (King et al. 1986). The outcome was the development of an amplification technique for detecting mRNA termed reverse transcriptase PCR (RT-PCR). To date however, due to the variable persistence of nucleic acids in cells post-death, the correlation between the presence of DNA and RNA and viability is still not clear (Cenciarini-Borde et al. 2009). In RT-PCR, an RNA strand is first reverse transcribed into its DNA complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is then amplified using conventional, multiplex, or real-time PCR. For example, Rijpens et al. (2002) targeted the housekeeping *rpoD* gene of *Salmonella.*  Overall, the assay could not detect viable *Salmonella* in heat or ethanol killed *Salmonella* cells. However, conventional RT-PCR techniques are labour intensive since the amplicon can be visualized only after the amplification ends, requires the rapid extraction of RNA due to its short half-life, suffers from an increased cross-contamination risk of the samples thus requiring DNase treatments, and the target genes must demonstrate abundant transcript expression, expression throughout the growth cycle, and negligible or no transcriptional regulation (Klein and Juneja 1997; Deisingh and Thompson 2004; Yaron and Matthews 2002). Due to these difficulties, the development of RT-PCR applications focusing on the detection of food-borne pathogens, including *Salmonella* in foods and environmental samples has been limited. D'Souza et al. (2009) developed a RT-PCR for the rapid detection of *Salmonella* using *invA* primers. Park et al. (2011) evaluated immunomagnetic beads and a RT-PCR method for the detection of *Salmonella* inoculated into poultry feed demonstrating that the *hilA* gene is a candidate for use in RT-PCR. Techathuvanan and D'Souza (2011) optimized a rapid *Salmonella* detection assay in liquid whole eggs by SYBR® Green based real-time RT-PCR targeting the *invA* gene as described previously for the detection of

of pathogens (Li and Mustapha 2004; Park et al. 2006).

**4.4 Reverse transcriptase PCR (RT-PCR)** 

Endpoint PCR is commonly utilized for the detection of amplified PCR products. However, DNA hybridization has also been described for detection (Chan et al. 1988; Hill and Keasler 1991; Hill and Lampel 1990). Probes directed to specific gene regions of the *Salmonella*  genome provide a powerful tool for use in DNA hybridization assays. Such methods of detection have proven to be more sensitive than agarose gel electrophoresis and more specific than culture or immunological based assays (Ten Bosch et al. 1992; Manzano et al. 1998). For example, Maciorowski et al. (1998) was able to detect PCR products from *S.*  Typhimurium inoculated animal feeds by hybridization with biotin and fluorescently labeled probes. Such specificity eliminates the need for serological confirmation and incidences of false-positive identification caused by antibody cross-reactivity with other organisms. Also, unlike biochemical differentiations, probe reactions do not rely on enzymatic activities and are therefore unaffected by media interference or the presence of bacteria with similar phenotypes. The majority of DNA based hybridization assays have exploited this specificity for DNA microarray assay targeting multiple genes with few applications related to the detection of *Salmonella* from food and environmental samples. Probes complimentary to amplified gene products have been used for the detection of *Salmonella* in oysters and chicken meat as well as from environmental poultry house drag swabs (Cohen et al. 1994; Doran et al. 1994; Jones et al. 1993; Bej et al. 1996). Commercial hybridization assays for the detection of *Salmonella* include the GeneQuence *Salmonella* assay (Neogen) utilizing probes previously evaluated by D'Aoust et al. (1995). This test employs Salmonella-Specific DNA probes, which are directly labeled with horseradish peroxidase. A colorimetric endpoint is then used for the detection of *Salmonella* spp. in food samples following broth culture enrichment with results available within 24 h.
