Developing A Real-Time PCR assay for detection of multi-drug resistant Salmonella strains in pigs

The main objective of this study is to develop a rapid, sensitive and accurate real-time detection assay for multi-drug resistant (MDR) Salmonella strains isolated from pigs. Standardization and preliminary testing of the assay is described as well as future directions for this research. 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.

Once standardization was complete, bacterial lysates and purified DNA samples were prepared, serially diluted, and analyzed using amplification and melt curves. In addition, standard curves were used to confirm the inverse relationship of concentration with amplification cycles and to determine the lowest concentration that generated the most sensitive and specific amplification. Currently, non-DT104/DT193 and non-Typhimurium strains are being tested and unique phage type primers are being designed to assess and enhance this assay (as demonstrated in the figure) for potential use in the field to prevent food-borne illness and track zoonotic pathogens.

Personnel:

Rebecca C. Robbins (PhD Student)
Wondwossen A. Gebreyes