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.