Development of a Real-time PCR Assay to Specifically Detect Salmonella Typhimurium

Introduction: In EU, Salmonella Typhimurium was recently deemed as an adulterant in poultry meat. While the current Kauffmann-White (K-W) serotyping method requires 4-5 days to assess the presence of the pathogen, a real-time PCR method could allow its specific detection within two days either as a rapid screening method or as a typing step after the detection of Salmonella spp. However, due to the close genetic relatedness between Salmonella serotypes, finding a unique target allowing distinguishing between them using a straightforward PCR assay is a challenging task.

Purpose: The purpose of this study was to identify and assess the value of a molecular target unique to Salmonella Typhimurium in order to detect this serotype specifically.

Methods: Height full Salmonella Typhimurium genomes found in the Genbank database were selected as targets and 21 non Salmonella Typhimurium genomes were selected as non-target sequences. Whole genome alignment and subtraction of regions of similarity was performed using ssGenfinder algorithm. Resulting sequences were then screened against the Salmonella WGS Genbank database and sequences yielding no cross-reactions with other serotypes were selected. Real-time PCR assay were designed and tested on a library of Salmonella Typhimurium and non-Typhimurium Salmonella serotypes.

Results: Two unique sequences were identified as being characteristic of the targeted Salmonella Typhimurium genomes. A simplex PCR assay was designed which showed 100% inclusivity on all Salmonella Typhimurium strains tested including monophasic variant and >99% on 254 non-Typhimurium Salmonella strains. Only one Salmonella Paratyphi B strain cross-reacted but not the Java variant.

Significance: These results suggest that it is possible to identify Salmonella Typhiumurium based on a unique sequence thus making real-time PCR assay an interesting alternative method for detecting this serotype. Furthermore, the strategy employed for seeking this unique sequence may be applied to other serotypes of interest in the food industry.