**5. Conclusions and perspectives**

364 Salmonella – A Dangerous Foodborne Pathogen

During the last decade, a number of studies have demonstrated the practicality of identifying *Salmonella* serovars using multiplex PCR (mPCR) (Kim et al., 2006). In addition, the technique has been shown to be a powerful and cost-effective tool for *Salmonella*  serotyping. For these reasons, we optimize a mPCR protocole to type the most common *Salmonella enterica* subsp. *enterica* serovars. This method is based on detection of genes present in specific serotypes. These genes were selected from analysis of previous work

The first step is to extract bacterial DNA. In this study, it was prepared by boiling (Agarwal et al. 2002). Then, we prepared the final PCR volume (34μl) that included: dNTPs mixture (0.2 mM); MgCl2 (2 mM); TaqDNA polymerase (5.0 units); primer(s) (50 ng each); genomic

All assays used the same cycling parameters under the following conditions: enzyme activation at 94°C for 5 min and then an additional 40 cycles with heat denaturation at 94°C for 30 s, primer annealing at 62°C for 30 s, and DNA extension at 72°C for 1 min. After the last cycle, samples were maintained at 72°C for 5 min to complete the synthesis of all

The PCR products (10μl) were separated by electrophoresis on 2% Tris-acetate EDTA agarose gel stained with ethidium bromide, visualized with UV induced fluorescence, and

The first multiplex PCR for *Salmonella* serotyping was applied using five primer sets in the same reaction mixture. Using these five STM primers with the 19 *Salmonella* serovars, we can identify four distinct groups (Imen et al. 2010). In a second approach, we validated the mPCR for *Salmonella* serovars detection by using STY primers. Thus, the 19 different tested *Salmonella* serovars could be classified into three groups on the basis of scoring the presence or absence of appropriately size amplicons (Imen et al. 2010). To further evaluate the discriminatory method for *Salmonella* serotyping and to increase identified serovars, we

In this study, using suitable primers for the two five-plex PCRs methods for molecular *Salmonella* serotyping, we could easily discriminate all the tested *Salmonella* serotypes that represented 100% of all *Salmonella* isolates in our laboratory. Also, a high rate of correlation was found between traditional and molecular serotyping. However, one exception was

These results have been found elsewhere (Perch et al. 2003). Whereas, we have noted a resemblance in molecular amplicon code in some *salmonella* serovars that can be explained by the presence of a very similar region in these serovars. It can also be explained by deletion problems that can concern a specific region and so the absence of appropriately sized amplicons with specific primers (Garaizar et al. 2002). A secondary discrimination problem that was interesting to note was that for *Anatum* serovar more than one amplicon

To further discriminate each serovar, we can associate to this multiplex PCR serotyping the PFGE analysis, or the 16 S\23 S r RNA ribotyping. These methods provided a high degree of

In this way, we describe the mPCR as a rapid, specific, and cost-effective molecular method that has demonstrated its efficient discrimination in serotyping of the most common clinical

combined molecular results of both the STM and STY primers (Imen et al. 2010).

found with *Salmonella Anatum* serotype (Imen et al. 2010).

code can be detected which may reflect intraserovar variation.

DNA template (5μl) and deionised water to make up the volume (Imen et al. 2010).

**Practical test of multiplex PCR method: Application and results in** *Salmonella* 

including whole-genome sequencing (Porwollik et al., 2004, 2005).

**serotyping** 

strands.

photographed (Imen et al. 2010).

intraserovar discrimination.

Overall the *Salmonella* demonstrate significant phenotypic diversity. Several phenotypic typing techniques have been developed and have been used successfully for decades. Over the years, serotype and phage type analyses have been particularly useful as evidenced by the success of the National *Salmonella* Surveillance System, and many other national surveillance projects throughout the world. However, these techniques have often been relegated to reference laboratories making rapid analysis by an individual laboratory difficult. An ideal typing method should fulfil the following six criteria: typeability, reproducibility, discriminatory power, and ease of interpretation, easy to use, and low cost. It is clear, that any method used currently for typing of *Salmonella* strains is an ideal method alone in terms of these criteria, but all methods exhibit benefits and also limitations. It is obvious that it is difficult to find a single method, which is most suitable for typing of *Salmonella* strains. As a consequence, the best discrimination has resulted from combinations of techniques, often a combination of phenotypic and genotypic techniques. At this time, major reference institutions rely on serotype analysis followed by PFGE as the gold standard for strain discrimination. PCR-based techniques, though, are more rapid and within a particular laboratory can be used as a primary screening tool for strain discrimination. Better standardization between laboratories will be required before any of the PCR techniques can become the method of choice. Additionally, validation in outbreak situations involving varied serotypes will be required to prove these techniques effective in the field.
