Abstract
BackgroundA fast and simple two-step multiplex real-time PCR assay has been developed to replace the traditional, laborious Salmonella serotyping procedure. Molecular beacons were incorporated into the assay as probes for target DNA. Target sequences were regions of the invA, prot6E and fliC genes specific for Salmonella spp. Salmonella Enteritidis and Salmonella Typhimurium, respectively, the two most clinically relevant serotypes. An internal amplification positive control was included in the experiment to ensure the optimal functioning of the PCR and detect possible PCR inhibition. Three sets of primers were used for the amplification of the target sequences. The results were compared to those of the Kauffmann-White antigenic classification scheme.ResultsThe assay was 100% sensitive and specific, correctly identifying all 44 Salmonella strains, all 21 samples of S. Enteritidis and all 17 samples of S. Typhimurium tested in this work. Therefore, the entire experiment had specificity and sensitivity of 100%. The detection limit was down to 10 copies of DNA target per 25 μl reaction.ConclusionThe assay can amplify and analyse a large number of samples in approximately 8 hours, compared to the 4 to 5 days conventional identification takes, and is thus considered a very promising method for detecting the two major serotypes of Salmonella quickly and accurately from clinical and environmental samples.
Highlights
A fast and simple two-step multiplex real-time PCR assay has been developed to replace the traditional, laborious Salmonella serotyping procedure
Thermal denaturation characteristics of molecular beacons Normalised fluorescence signals for both the beacon and the beacon-target hybrid were plotted against temperature to give a thermal denaturation profile for each beacon (Fig. 1)
Complementary beacon-target hybrids exist at lower temperatures giving out a bright fluorescence signal
Summary
A fast and simple two-step multiplex real-time PCR assay has been developed to replace the traditional, laborious Salmonella serotyping procedure. Target sequences were regions of the invA, prot6E and fliC genes specific for Salmonella spp. Salmonella is a gram-negative, facultative anaerobic, flagellated bacterium. It is the pathogenic agent of salmonellosis, a major cause of enteric illness and typhoid fever, leading to many hospitalisations and a few rare deaths if no antibiotics are administered. The detection of Salmonella remains a highly important issue in microbiological analysis for food safety and standards. Enterica is the most clinically significant, causing 99% of Salmonella infections. In the present study we are concerned with its two main serovars: Salmonella enterica serovar Typhimurium (group D) denoted S. Typhimurium, and Salmonella enterica serovar Enteritidis (group B) denoted S. Enteritidis, which are the most commonly isolated Salmonellae from food-borne outbreaks
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