In Silico-Based Development of a Sensitive PCR Assay to Detect and Differentiate three major Salmonella enterica Serovars

Abstract

Serovars of the bacterium Salmonella enterica are important worldwide causative agents of bacterial food poisoning. Of more than 1500 serovars of this species; Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Gallinarum are the most important serovars affecting human health and poultry industry. They cause great economic losses both due to their implication in animal welfare and their ability to contaminate poultry food products forming a serious food safety problem. Early detection and typing of these serovars is essential for food safety regulations and poultry industry. The current techniques to detect and serotype these serovars are time consuming and require technical expertise. The aim of this work was to develop a practical PCR-based method that can detect and distinguish the above mentioned 3 serovars. Genomic sequence data of Salmonella Gallinarum, Salmonella Enteritidis and Salmonella Typhimurium was used through NCBI database (as of Month 2015). Intensive comparative genomic analysis was conducted to identify unique segments for each serovar. The identified unique segments were used as specific markers to design 3 close nested PCR assays that can detect and distinguish the three serovars. The detection specificity of the PCR assays were tested using a wide range of Salmonella enterica serovars. The detection sensitivity of the three PCR tests was examined using 10 fold serial dilutions of the control DNAs. The analysis revealed unique segments for each serovar that can serve as specific genetic markers. PCR-primers for each unique segment produced the specific amplicon for each serovar. The developed close nested PCR techniques showed amplification signal of as low as (0.03 ng- 3 pg) DNA, which is 100-fold more sensitive than traditional PCR protocols. Three of the most important Salmonella enterica serovars, which are associated with poultry health and human food poisoning were detected and typed by a very sensitive PCR technique. The developed PCR can be used as a routine test to manage and control the poultry health and safety of poultry products.