High-throughput multiplex qPCR for distinguishing Salmonella serovars Heidelberg, Enteritidis, Typhimurium, Gallinarum, and Pullorum.

Brazilian regulation under the National Salmonella Control Program mandates surveillance of serovars Enteritidis, Typhimurium, Gallinarum, and Pullorum in commercial broiler chicken and turkey farms. However, the recent emergence of Salmonella Heidelberg in poultry products highlights a new concern regarding the spread of additional serovars. In this context, the present study aims to standardize a multiplex qPCR assay capable of simultaneously differentiating the four regulated serovars and S. Heidelberg, optimizing sensitivity and analysis speed to effectively address current sanitary challenges in poultry production. Salmonella isolates were obtained from the microbiological collections of the Serviço de Orientação à Alimentação Pública (SOAP), the Serviço de Ornitopatologia FMVZ/UNESP, and from SOAP’s routine laboratory. Bacterial genomic DNA was purified using an internal protocol based on magnetic beads, and genus-level detection of Salmonella was performed by real-time PCR (qPCR) targeting a conserved region of the invA gene. For the multiplex assay, primers and probes specific to each regulated serovar were employed according to previous standardizations, while the probe specific for S. Heidelberg was designed in silico and experimentally validated within this study. The multiplex qPCR reaction was optimized using five combinations of primer concentrations (0.05–0.30 µM) and probe concentrations (0.05–0.15 µM). The limit of detection was established from serial dilutions of genomic DNA, and the assay was validated on 21 typified S. Heidelberg strains, followed by evaluation on 41 Salmonella isolates from naturally contaminated samples. All tested conditions yielded consistent amplification of S. Heidelberg isolates, and the mix containing primers at 0.10 µM and probe at 0.05 µM was selected as the most robust. The limit of detection (LOD) was defined to a maximum Cq value of 33.39. When evaluating 21 typified S. Heidelberg strains using the standardized multiplex assay, Cq values ranged from 16.03 to 27.06 (mean = 18.55; median = 17.36; standard deviation = 2.79). In a set of 41 isolates from naturally contaminated samples, four exhibited amplification for the S. Heidelberg target, with Cq values of 18.04, 17.60, 16.00, and 16.61. The standardized multiplex qPCR assay consistently detected S. Heidelberg and regulated serovars. This rapid, robust, and adaptable diagnostic meets the poultry industry’s need for accurate, timely detection, enabling early surveillance of emerging serovars like S. Heidelberg and supporting active monitoring and risk mitigation.