**4.2 Real-time PCR**

The development of novel chemistries and instrumentation platforms enabling detection of PCR products on a real-time basis has led to widespread adoption of real-time PCR as the method of choice for detection of *Salmonella* (Espy et al. 2006). This method combines amplification and detection stages of the process so that nucleic acid amplification is monitored and recorded continuously hence eliminating the need for post-amplification steps such as gel electrophoresis. The detection of PCR products is accomplished via the generation of a fluorescent signal by any of the commercially available chemistries for realtime PCR: TaqMan® (Applied Biosystems®), Molecular Beacons, Scorpions®, and SYBR® Green (Molecular Probes), among others.

The simplest approach involves the use of the intercalating fluorescent dye SYBR® Green. This fluorogenic dye exhibits little fluorescence when in solution, but emits a strong

*Salmonella* Detection Methods for Food and Food Ingredients 381

hour commercial real time PCR assay for the detection of *S.*Enteritidis was developed by

In multiplex PCR (mPCR), several specific primer sets are combined into a single PCR assay for the simultaneous amplification of more than one target DNA sequence (Chamberlain et al. 1988). As with conventional or endpoint PCR, the amplified DNA targets are separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Depending on the number of targets, the analysis is carried out by a single amplification reaction of four–to five targets, or could take place via a two-step amplification reaction for five–six targets or more (Settanni and Corsetti 2007). For example, Malorny et al. (2007) developed an assay for the specific detection of *S.* Enteritidis in whole chicken carcass rinses and consumption eggs. The assay used specifically designed primers and a TaqMan probe to target the *Prot6e* gene located on the *S*. Enteritidis 60-kb virulence plasmid. As an internal amplification control to monitor *Salmonella* DNA in the sample, a second primer/TaqMan probe set detected simultaneously the *Salmonella* specific *invA* (invasion protein A) gene. It must be considered however, that the majority of the articles in the scientific literature deal with mPCR methods developed to identify and or characterize *Salmonella* serotypes from pure cultures, or in controlled artificial inoculation experiments, with only a minority of studies providing results from *in situ* detection of pathogens in foods or environmental samples. Soumet et al. (1999) developed a multiplex PCR assay for the simultaneous identification of *Salmonella* species, *S.* Enteritidis and *S.* Typhimurium from environmental swabs of poultry houses. Similarly, O'Regan et al. (2008) developed a real-time multiplex PCR assay for the detection of multiple *Salmonella* serotypes in chicken samples. Poultry-associated serotypes detected in the assay included *S*. Enteritidis, *S*. Gallinarum, *S*. Typhimurium, *S*. Kentucky and *S*. Dublin. Generally, the 16S rRNA gene is the most common target for mPCR as it is routinely used to establish phylogenetic distinctions among bacteria (Rossello-Mora and Amman 2001). However, other target genes are also considered in order to achieve a high specificity. For example, Rajtak et al. (2011) developed a two step real-time mPCR assay for the rapid screening of 19 *Salmonella* serotypes frequently encountered in humans, animals, and animal-associated meat products within the European Union. Specific primers for serotype differentiation were designed to target the genes encoding either phase 1 and 2 flagellar antigens *fliC* and *fljB* or unique serotype-specific loci. In addition, the assay simultaneously screened for the presence of the ampicilin-amoxicillin, chloramphenicol-florfenicol, streptomycin-spectinomycin, sulfanomides, and tetracycline (ACSSuT)-type multidrug resistance pattern, indicated by the *floR* gene, and for the *Salmonella* virulence plasmid encoded by the svp operon in *S.* Typhimurium. The assay represents a more rapid and reliable method for identification of large numbers of serotypes than assays using phenotypic serotyping methods. Multiplex PCR is thus quite versatile and numerous other assays have been published for the rapid detection and characterization of specific *Salmonella* serotypes (Alvarez et al. 2004; Woods et al. 2008; Kim et al. 2006; Chiu et al. 2006) analogous to mPCR approaches used for the differentiation of multiple species belonging to single genera such as gastroenteritis causing themotolerant *Campylobacter* species (Korolik et al. 2001;Klena et al. 2004; Wang et al. 2002; Yamazaki-Matsune et al. 2007) or for the differentiation of the major *L. monocytogenes* serovars (1/2a, 1/2b, 1/2c, and 4b) commonly implicated in food borne listeriosis (Doumith et al. 2004; Zhang and Knabel 2005; Chen and

Applied Biosystems®, the TaqMan® *Salmonella* Enteritidis Detection Kit.

**4.3 Multiplex PCR** 

fluorescent signal upon binding to double-stranded DNA. Thus, as a PCR product accumulates, fluorescence increases. The advantages of SYBR® Green are that it is inexpensive, simple, and sensitive. The disadvantage is that SYBR® Green will bind to any double-stranded DNA in the reaction, which may result in an overestimation of the target concentration. A second, more accurate and reliable method is to use fluorescent reporter probes (TaqMan®, Molecular Beacons, Scorpions®). These probes depend on Förster Resonance Energy Transfer (FRET) to generate the fluorescence signal via the coupling of a fluorogenic dye molecule and a quencher moeity to the same or different oligonucleotide substrates. The main advantage of TaqMan probes, Molecular Beacons and Scorpions is that they allow for multiplex PCR assays by using spectrally separated fluor/quench moieties for each probe. Multiplex PCR allows internal controls to be co-amplified and permits allele discrimination in single-tube, homogeneous assays. These hybridization probes afford a level of discrimination impossible to obtain with SYBR® Green, since they will only hybridize to true targets in a PCR and not to primer-dimers or other spurious products. However these probes can be expensive to synthesize, with a separate probe needed for each target being analyzed.

Commercial real time PCR assays employ a high degree of automation to reduce the number of operations involved and reduce the risk of contamination. The reaction usually takes place inside a combined thermocycler-fluorescence detection instrument and uses preprepared reagents, often in a dehydrated tablet form. The thermo-cycling and detection are controlled by software that also calculates and interprets the results. Total time for an analysis for the detection of *Salmonella* species is normally 20 to 48 hours but can be as little as 12 hours depending on the food matrix, enrichment conditions, and instrument run time. The main advantage of these PCR systems over other methods is in time saving, both in the total time from sampling to result and in the technical time needed to set up and run the assay. In addition many available real time PCR assays have achieved a variety of certifications via AOAC, AFNOR, NORDVAL, and ISO 16140 validation. However, capital costs for automated PCR systems are relatively high and consumable costs are also high by comparison to culture based techniques. There is a clear cost benefit in rapid test results allowing faster HACCP verification and release of finished food products particularly where the prevalence of *Salmonella* is known to be low, thus reducing additional culture confirmation tests or where pooling of samples is permitted. Numerous assays are commercially available using real time PCR for the detection of *Salmonella.* The BAX PCR detection system (DuPont-Qualicon Inc.), a platform adopted by USDA-FSIS as a screening tool offers a detection kit for the detection of *Salmonella* in a variety of food and food ingredients. Other systems offering similar testing capabilities include: ADIAFOOD Rapid Pathogen Detection System (AES Chemunex), the Assurance Genetic Detection System GDS (Biocontrol Inc.) utilizing a post enrichment IMS step followed by real time PCR, iQ-Check™ Salmonella II (BioRad Laboratories, S.A.), and R.A.P.I.D. LT system (Idaho Technology Inc.), among others.

Lastly, real time PCR systems have sufficient flexibility to allow for the rapid development of new assays targeting specific *Salmonella* serovars of clinical significance. More recently in 2010, in order to minimize the potential for foodborne illness from eggs containing *S.*  Enteritidis, the FDA implemented new regulations for the egg industry, which included requiring large-scale egg producers to begin SE monitoring programs in their poultry houses and potentially on their products. In response to the industry testing needs, a 27 hour commercial real time PCR assay for the detection of *S.*Enteritidis was developed by Applied Biosystems®, the TaqMan® *Salmonella* Enteritidis Detection Kit.

#### **4.3 Multiplex PCR**

380 Salmonella – A Dangerous Foodborne Pathogen

fluorescent signal upon binding to double-stranded DNA. Thus, as a PCR product accumulates, fluorescence increases. The advantages of SYBR® Green are that it is inexpensive, simple, and sensitive. The disadvantage is that SYBR® Green will bind to any double-stranded DNA in the reaction, which may result in an overestimation of the target concentration. A second, more accurate and reliable method is to use fluorescent reporter probes (TaqMan®, Molecular Beacons, Scorpions®). These probes depend on Förster Resonance Energy Transfer (FRET) to generate the fluorescence signal via the coupling of a fluorogenic dye molecule and a quencher moeity to the same or different oligonucleotide substrates. The main advantage of TaqMan probes, Molecular Beacons and Scorpions is that they allow for multiplex PCR assays by using spectrally separated fluor/quench moieties for each probe. Multiplex PCR allows internal controls to be co-amplified and permits allele discrimination in single-tube, homogeneous assays. These hybridization probes afford a level of discrimination impossible to obtain with SYBR® Green, since they will only hybridize to true targets in a PCR and not to primer-dimers or other spurious products. However these probes can be expensive to synthesize, with a separate probe needed for

Commercial real time PCR assays employ a high degree of automation to reduce the number of operations involved and reduce the risk of contamination. The reaction usually takes place inside a combined thermocycler-fluorescence detection instrument and uses preprepared reagents, often in a dehydrated tablet form. The thermo-cycling and detection are controlled by software that also calculates and interprets the results. Total time for an analysis for the detection of *Salmonella* species is normally 20 to 48 hours but can be as little as 12 hours depending on the food matrix, enrichment conditions, and instrument run time. The main advantage of these PCR systems over other methods is in time saving, both in the total time from sampling to result and in the technical time needed to set up and run the assay. In addition many available real time PCR assays have achieved a variety of certifications via AOAC, AFNOR, NORDVAL, and ISO 16140 validation. However, capital costs for automated PCR systems are relatively high and consumable costs are also high by comparison to culture based techniques. There is a clear cost benefit in rapid test results allowing faster HACCP verification and release of finished food products particularly where the prevalence of *Salmonella* is known to be low, thus reducing additional culture confirmation tests or where pooling of samples is permitted. Numerous assays are commercially available using real time PCR for the detection of *Salmonella.* The BAX PCR detection system (DuPont-Qualicon Inc.), a platform adopted by USDA-FSIS as a screening tool offers a detection kit for the detection of *Salmonella* in a variety of food and food ingredients. Other systems offering similar testing capabilities include: ADIAFOOD Rapid Pathogen Detection System (AES Chemunex), the Assurance Genetic Detection System GDS (Biocontrol Inc.) utilizing a post enrichment IMS step followed by real time PCR, iQ-Check™ Salmonella II (BioRad Laboratories, S.A.), and R.A.P.I.D. LT system (Idaho

Lastly, real time PCR systems have sufficient flexibility to allow for the rapid development of new assays targeting specific *Salmonella* serovars of clinical significance. More recently in 2010, in order to minimize the potential for foodborne illness from eggs containing *S.*  Enteritidis, the FDA implemented new regulations for the egg industry, which included requiring large-scale egg producers to begin SE monitoring programs in their poultry houses and potentially on their products. In response to the industry testing needs, a 27

each target being analyzed.

Technology Inc.), among others.

In multiplex PCR (mPCR), several specific primer sets are combined into a single PCR assay for the simultaneous amplification of more than one target DNA sequence (Chamberlain et al. 1988). As with conventional or endpoint PCR, the amplified DNA targets are separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Depending on the number of targets, the analysis is carried out by a single amplification reaction of four–to five targets, or could take place via a two-step amplification reaction for five–six targets or more (Settanni and Corsetti 2007). For example, Malorny et al. (2007) developed an assay for the specific detection of *S.* Enteritidis in whole chicken carcass rinses and consumption eggs. The assay used specifically designed primers and a TaqMan probe to target the *Prot6e* gene located on the *S*. Enteritidis 60-kb virulence plasmid. As an internal amplification control to monitor *Salmonella* DNA in the sample, a second primer/TaqMan probe set detected simultaneously the *Salmonella* specific *invA* (invasion protein A) gene. It must be considered however, that the majority of the articles in the scientific literature deal with mPCR methods developed to identify and or characterize *Salmonella* serotypes from pure cultures, or in controlled artificial inoculation experiments, with only a minority of studies providing results from *in situ* detection of pathogens in foods or environmental samples. Soumet et al. (1999) developed a multiplex PCR assay for the simultaneous identification of *Salmonella* species, *S.* Enteritidis and *S.* Typhimurium from environmental swabs of poultry houses. Similarly, O'Regan et al. (2008) developed a real-time multiplex PCR assay for the detection of multiple *Salmonella* serotypes in chicken samples. Poultry-associated serotypes detected in the assay included *S*. Enteritidis, *S*. Gallinarum, *S*. Typhimurium, *S*. Kentucky and *S*. Dublin. Generally, the 16S rRNA gene is the most common target for mPCR as it is routinely used to establish phylogenetic distinctions among bacteria (Rossello-Mora and Amman 2001). However, other target genes are also considered in order to achieve a high specificity. For example, Rajtak et al. (2011) developed a two step real-time mPCR assay for the rapid screening of 19 *Salmonella* serotypes frequently encountered in humans, animals, and animal-associated meat products within the European Union. Specific primers for serotype differentiation were designed to target the genes encoding either phase 1 and 2 flagellar antigens *fliC* and *fljB* or unique serotype-specific loci. In addition, the assay simultaneously screened for the presence of the ampicilin-amoxicillin, chloramphenicol-florfenicol, streptomycin-spectinomycin, sulfanomides, and tetracycline (ACSSuT)-type multidrug resistance pattern, indicated by the *floR* gene, and for the *Salmonella* virulence plasmid encoded by the svp operon in *S.* Typhimurium. The assay represents a more rapid and reliable method for identification of large numbers of serotypes than assays using phenotypic serotyping methods. Multiplex PCR is thus quite versatile and numerous other assays have been published for the rapid detection and characterization of specific *Salmonella* serotypes (Alvarez et al. 2004; Woods et al. 2008; Kim et al. 2006; Chiu et al. 2006) analogous to mPCR approaches used for the differentiation of multiple species belonging to single genera such as gastroenteritis causing themotolerant *Campylobacter* species (Korolik et al. 2001;Klena et al. 2004; Wang et al. 2002; Yamazaki-Matsune et al. 2007) or for the differentiation of the major *L. monocytogenes* serovars (1/2a, 1/2b, 1/2c, and 4b) commonly implicated in food borne listeriosis (Doumith et al. 2004; Zhang and Knabel 2005; Chen and

*Salmonella* Detection Methods for Food and Food Ingredients 383

*Salmonella* from jalapeno and serrano peppers, and Pork (Miller et al. 2010; Techatuvanan et al. 2010). To further address the issue of viability of the species detected in a complex matrix such as foods, perhaps the best alternative could be the development and validation of real time and multiplexed PCR assays targeting mRNA, also termed multiplex RT-PCR (Gonzalez-Escalona et al. 2009; Settanni and Corsetti 2007). Thus far however, no commercial PCR assay is available utilizing reverse-transcriptase technology for detecting

It is evident that molecular methods offer improved sensitivity and potential reduction in assay time. It has now become possible to rapidly detect and confirm the presence of foodborne *Salmonella* spp. in a wide array of food and environmental samples by commercial amplification detection systems. The primary challenges remaining are to develop more reliable recovery and extraction procedures for routine processing of samples from a wider variety of feed and environmental matrices and apply molecular techniques

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.

Bacteriophages are viruses infecting bacteria and by definition obligate intracellular parasites lacking their own metabolism, are extremely host-specific, and able only to infect specific species or even strains. Virulent phages with a broad host range within the *Salmonella* genus are ideally suited for detection purposes since they are unable to integrate

**5. Phage based detection methods** 

*Salmonella* in foods.

for further characterizing *Salmonella* spp.

**4.5 Nucleic acid hybridization** 

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

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

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 *Salmonella* from jalapeno and serrano peppers, and Pork (Miller et al. 2010; Techatuvanan et al. 2010). To further address the issue of viability of the species detected in a complex matrix such as foods, perhaps the best alternative could be the development and validation of real time and multiplexed PCR assays targeting mRNA, also termed multiplex RT-PCR (Gonzalez-Escalona et al. 2009; Settanni and Corsetti 2007). Thus far however, no commercial PCR assay is available utilizing reverse-transcriptase technology for detecting *Salmonella* in foods.

It is evident that molecular methods offer improved sensitivity and potential reduction in assay time. It has now become possible to rapidly detect and confirm the presence of foodborne *Salmonella* spp. in a wide array of food and environmental samples by commercial amplification detection systems. The primary challenges remaining are to develop more reliable recovery and extraction procedures for routine processing of samples from a wider variety of feed and environmental matrices and apply molecular techniques for further characterizing *Salmonella* spp.
