Real time PCR gene profiling and detection of Salmonella using a novel target: The siiA gene

• Published in: Journal of microbiological methods 2015-02, Vol.109, p.9-15
• Main Authors: Ben Hassena, Amal, Barkallah, Mohamed, Fendri, Imen, Grosset, Noel, Ben Neila, Idriss, Gautier, Michel, Gdoura, Radhouane
• Format: Article
• Language: English
• Subjects: Animals; DNA Primers - genetics; Food and Nutrition; Gene Expression Profiling - methods; Humans; Life Sciences; Microbiology and Parasitology; Real time PCR; Real-Time Polymerase Chain Reaction; Salmonella; Salmonella - classification; Salmonella - genetics; Salmonella - isolation & purification; Salmonella detection; Salmonella Infections - diagnosis; Salmonella Infections - microbiology; Salmonella Infections, Animal - diagnosis; Salmonella Infections, Animal - microbiology; SPI; spvC; Transition Temperature; Virulence
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2014.11.018

The objective of this study was to develop and evaluate a SYBR Green real time PCR method for the specific detection of Salmonella spp using a novel target, the siiA gene. Primer specificity testing was done on a panel of 76 Salmonella strains and 32 non-Salmonella strains. The primers directed against the siiA gene amplified all Salmonella strains tested, while non-Salmonella strains were not amplified. The melting temperatures of the 107bp amplicons were consistently specific as they gave melting peaks around 75.5°C. The precision of the assay, based on intra and inter-run variations, was shown to be widely acceptable. In the second part of this study, 45 Salmonella strains were screened for the presence of 6 virulence-associated genes (sopB, cat2, safC, sefB and SC1248) located in several Salmonella Pathogenicity Islands (SPIs) and the spvC gene from the Salmonella virulence plasmid. The prevalence of these genes ranged from 51% to 100%. Variable virulence gene profiles were obtained even within the same serotype. •We have successfully optimized a real time PCR for Salmonella detection in food using novel primers based on the siiA gene.•This real-time PCR is an excellent alternative to conventional methods for diagnostic purposes and official surveillance.•Variability in virulence profiles reflecting an important genetic variability.•Real time PCR could serve as a genotyping method as an alternative to high costing and sophisticated techniques