Two five-plex PCRs methods for identification of common Salmonella spp. serotypes

Abstract

In Tunisia, Salmonella is the most common bacterial agent responsible for childhood diarrhoea. Currently, isolation of the bacterium by microbiological and biochemical methods and confirmation of the serotype by serological method remain as the "gold standard". This study aimed to differentiate among the most common serotypes of Salmonella spp. via two rapid five-plex PCRs assay (MPCR) to evaluate the molecular serotyping method compared with the gold standard serotyping technique. The two five-plex PCRs assays were designed for the simultaneous detection of six genetic loci from Salmonella enterica serovar Typhimurium and four from S. enterica serovar Typhi. Sixty-one Tunisian strains (46 collected from patients and 15 from food) were isolated during the period 2002-2007. The STM and STY primers were able to discriminate all tested Salmonella serotypes that represent the most common clinical and food strains of S. enterica subsp. enterica in our laboratory. All strains belonged to 19 different serotypes: 15 serotypes gave unique amplification patterns compared each other and the other 4 serotypes were grouped into two pairs that gave the same molecular profile. We resolved this problem through the addition of a monoplex PCR. Salmonella typhimurium ATCC 14028 consistently produced the same molecular profile as S. typhimurium laboratory isolates. Interestingly, seven strains of Anatum serovar produced two different PCR profiles with these primers: five strains had the same amplification pattern STM 2,4,5 and STY 2; however, two strains had another molecular profile STM 2,3,5 and STY 2; so the reproducibility of this method was reduced to 93%. The MPCR system is a rapid, specific, and cost-effective molecular method that gas been proved to have efficient discrimination in serotyping of the most common isolates of S. enterica subsp. enterica.

Laboratory Typing Methods for Diagnostic of Salmonella Strains, the “Old” Organism That Continued Challenges

“…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.…”

“…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.…”

“…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).…”

“…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.…”