Propidium monoazide real-time PCR amplification for viable Salmonella species and Salmonella Heidelberg in pork

Salmonella enterica serovar Heidelberg causes foodborne infections and is a major threat to the food chain and public health. In this study, we aimed to develop a rapid molecular typing approach to identify Salmonella enterica serovar Heidelberg. Using comparative genomics, four serovar-specific gene fragments were identified, and a real-time polymerase chain reaction (PCR) combined with a propidium monoazide (PMA) pretreatment method was developed for simultaneous detection of viable Salmonella sp. (invA) and Salmonella Heidelberg (SeHA_ C3258). The assay showed 100% specificity for all strains tested. The assay was able to distinguish effectively viable or dead cells with the PMA. The detection limit was 2.4 CFU/mL following 6 h of incubation in enrichment Luria-Bertani medium, and the assay could detect 1.7 x 10(2) CFU/mL in the presence of pork background flora. In artificially contaminated pork, real-time PCR detected inoculum levels of 1.15 CFU/25 g of pork after a 6 h enrichment. Thus, our findings indicated that this comparative genomics approach could be used to screen for serovar-specific fragments and that real-time PCR with PMA was a simple and reliable method for detecting viability of Salmonella species and Salmonella Heidelberg.