Development and validation of a genome-informed multiplex PCR for specific detection of typhoidal Salmonella serovars

Enteric fever caused by Salmonella enterica serovars Typhi and Paratyphi A, B and C remains a major public health burden in endemic regions. Existing molecular assays frequently demonstrate limited specificity due to cross-reactivity with non-typhoidal Salmonella (NTS). In this study, we developed and validated a genomics-informed multiplex PCR assay capable of simultaneously differentiating all four typhoidal Salmonella serovars. A curated dataset of 3,239 Salmonella genomes, including S. Typhi (n=361), S. Paratyphi A (n=453), S. Paratyphi B (n=511), S. Paratyphi C (n=62), and NTS genomes (n=1,853), was used for comparative genomic analysis. Thirty published PCR targets were evaluated in silico, followed by pangenome and SNP analyses to identify discriminatory loci for mismatch amplification mutation assay (MAMA)-based primer design. Candidate primers were validated using in silico PCR, BLASTn analysis, and laboratory testing against a panel of typhoidal Salmonella, clinical NTS isolates, and non-Salmonella bacterial pathogens. In silico evaluation demonstrated substantial cross-reactivity among many published targets, whereas SNP-informed primer design targeting staG (S. Typhi), SPA0152 (S. Paratyphi A), SPAB_03490 (S. Paratyphi B), and SPC_0571 (S. Paratyphi C) achieved predicted specificities of 98-100% while retaining high analytical sensitivity (>97%) across target genomes. Combined with a pan-Salmonella invA target, the multiplex assay precisely identified all target serovars in vitro with minimal cross-reactivity. These findings demonstrate that genomics-informed SNP-based primer design enables reliable multiplex differentiation of typhoidal Salmonella serovars and provides a scalable framework for improving enteric fever diagnosis and surveillance in endemic settings. ImportanceTyphoidal Salmonella serovars remain major causes of enteric fever in endemic regions, yet molecular differentiation from non-typhoidal Salmonella (NTS) remains challenging because of extensive genomic conservation and cross-reactivity of commonly used diagnostic targets. In this study, we combined large-scale comparative genomics of 3,239 Salmonella genomes with SNP-informed primer design to develop a multiplex PCR assay capable of simultaneously differentiating all four typhoidal serovars (S. Typhi, S. Paratyphi A, B, and C) from NTS and other non-Salmonella pathogens. Unlike conventional gene-content-based assays, this approach incorporated lineage-specific SNPs and mismatch amplification strategies to improve specificity while maintaining high analytical sensitivity. In silico evaluation demonstrated high diagnostic performance across diverse global lineages, while in vitro testing confirmed accurate serovar-level discrimination with minimal cross-reactivity. These findings demonstrate the value of population-scale genomics for molecular assay development and provide a scalable framework for improving diagnosis and surveillance of enteric fever in endemic settings.