Development and Evaluation of a TaqMan Real-Time PCR Assay for the Detection of Salmonella spp. for Laboratory and Epidemiological Surveillance

Reliable detection of Salmonella is essential for public health surveillance and food safety, particularly given the diversity of circulating serovars and the potential limitations of individual molecular targets. This study comparatively evaluated six candidate targets (hilA, invA, phoP, fimA, rpoS, and spvA) and developed a hilA-targeting TaqMan real-time PCR assay for Salmonella detection. To the best of our knowledge, this is the first study in Kazakhstan to apply a comparative target-selection approach to the development of a hilA-based real-time PCR assay. Among the targets evaluated, hilA demonstrated the broadest amplification coverage across the tested isolate panel, including less common serovars, whereas invA was not amplified in a subset of iso-lates. The developed assay demonstrated limits of detection of 10² CFU/mL for bacterial suspensions and 10 fg/μL for genomic DNA, with an amplification efficiency of 93.4% and an R² of 0.9988 over the linear range from 1 ng/μL to 10 fg/μL. No cross-reactivity was observed within the tested non-target bacterial panel. All 24 whole-genome sequencing-confirmed Salmonella isolates yielded positive amplification within the predefined Ct threshold (≤37), including S. enterica serovar Dublin strain 25S, which showed the highest mean Ct value (36.66 ± 0.14). The Proteus mirabilis comparator, initially identified as Salmonella by conventional bacteriology, showed no specific amplification. Salmonella DNA was also detected in both artificially contaminated food matrices tested. These findings support hilA as a promising molecular target and demonstrate the potential of the developed assay for laboratory detection, food safety investigations, and epidemiological surveillance. Further validation using broader isolate collections, naturally contaminated samples, and independent laboratories is warranted.