Development and Analytical Evaluation of a hilA-Targeting TaqMan Real-Time PCR Assay for Salmonella enterica Detection

Rapid and reliable molecular detection of Salmonella is important for laboratory investigation and infectious disease surveillance. This study compared six candidate molecular targets (hilA, invA, phoP, fimA, rpoS, and spvA) and developed a hilA-targeting TaqMan real-time PCR assay for Salmonella enterica detection. During conventional PCR screening, the hilA-M1-F/R primer pair demonstrated the most consistent and reproducible amplification across the tested isolate panel under the experimental conditions used and was therefore selected for subsequent assay development. The developed assay showed an amplification efficiency of 93.4% and an R2 of 0.9988 over a linear genomic DNA range from 1 ng/μL to 10 fg/μL. Experimentally determined detection limits under the conditions tested were 102 CFU/mL for bacterial suspensions and 10 fg/μL for genomic DNA. All 24 whole-genome sequencing-confirmed S. enterica isolates representing multiple serovars and sequence types were reproducibly detected, whereas no specific amplification was observed within the expanded panel of 15 non-target bacterial strains or isolates. The assay also detected S. enterica DNA in artificially contaminated milk and carrot matrices in preliminary proof-of-concept experiments. These findings demonstrate promising analytical performance of the hilA-targeting TaqMan assay within the evaluated experimental framework. However, broader validation using geographically diverse S. enterica isolate collections, additional non-target organisms, internal amplification controls, naturally contaminated food products, primary clinical, veterinary, and environmental specimens, and independent laboratories is required before routine diagnostic, food-testing, or epidemiological surveillance applications can be established.