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

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

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

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 strands.

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 photographed (Imen et al. 2010).

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

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

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 code can be detected which may reflect intraserovar variation.

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 intraserovar discrimination.

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 and food isolates of *S. enterica* subsp. *enterica* in our region. This technique can be used as an alternative method of standard serotyping in many clinical laboratories.
