Developing real-time PCR assay for detection of multi-drug Salmonella strains in pigs

Abstract

The main objective of this study was to develop a rapid, sensitive and accurate real-time detection assay for multi-drug resistant (MDR) Salmonella strains isolated from pigs. Initially, standardized procedures for use with real-time PCR using SYBR ® green were developed to evaluate selected primers, detection limitations using two predominant strains: S. Typhimurium phage types DT104 and DT193. The use of bacterial lysate and purified DNA samples was also compared. Lysate dilutions for concentrations (100-10-5) resulted in widely varied amplification as reflected in amplification curves. In contrast, purified DNA samples diluted to 10-8 µg/mL resulted in lower threshold cycles thus was found to be a more reliable procedure. The low detection limit and therefore, increased sensitivity of real-time PCR suggests this technology could be the cornerstone for development of assays for potential use on farms, in slaughter facilities, and by veterinarians in field efforts to detect pathogens in biological samples before organisms cause foodborne illness in humans.