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Salmonella enterica serovar Paratyphi C is highly adapted to humans and can cause a typhoid-like disease with high mortality rates. In this study, three serovar-specific genes were identified by comparative genomics for Salmonella Paratyphi C, SPC_0871, SPC_0872, and SPC_0908. Based on the SPC_0908 and xcd genes for testing Salmonella spp., we developed a duplex real-time nucleic acid sequence-based amplification (real-time NASBA) with a molecular beacon approach for the simultaneous detection of viable cells of Salmonella spp. and serotype Paratyphi C. The test selectively and consistently detected 53 Salmonella spp. (representing 31 serotypes) and 18 non-Salmonella strains. Additionally, the method showed high resistance to interference from natural background flora in pork and chicken samples. The sensitivity of the established approach was determined to be 4.89 cfu/25 g in artificially contaminated pork and chicken samples after pre-enrichment. We propose this NASBA-based protocol as a potential detection method for Salmonella spp. and serotype Paratyphi C in foods of animal origin.

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 × 102 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.