## 4 Discussion
enterica_ subsp. _ enterica_ sv. Typhimurium vaccines, such as Primun Salmonella T, is considered an effective tool for reducing its prevalence in poultry flocks, thereby decreasing the risk of transmission along the food chain. Therefore, a rapid and reliable method to distinguish the vaccine strain from wild strains of _S. enterica_ subsp. _ enterica_ sv. Typhimuriumium is essential to ensure a diagnosis that can distinguish infected from vaccinated animals. GPSTM has previously developed a qPCR DIVA assay that allows effective discrimination of Primun Salmonella T vaccine and wild strains from _S. enterica_ subsp. _ enterica_ sv. Typhimuriumium colonies isolated following current normalised culturing. This qPCR assay, including a rapid DNA extraction step, provided accurate results in less than 40 min, whereas an antibiogram to identify strain ST CAL16 Str+/Rif+/Enr- may take 24 h or longer. This remarkable improvement in turnaround time facilitates timely decision-making for infection control measures. The specificity of the SaITypm and PriSal-T assays was evaluated in vitro using reference genomic material. The SaITypm assay was specific for the detection of _S. enterica_ subsp. _enterica_ sv. Typhimuriumium, including the vaccine strain. Conversely, the PriSal-T assay specifically detected the vaccine strain but not wild _S. enterica_ subsp. _enterica_ sv. Typhimuriumium strains. The SaITypm&PriSal-T qPCR Duplex kit was subjected to analytical validation following the guidelines recommended by the international standard ISO/IEC 17025:2017 and the results obtained were optimal according to the ranges established as acceptance criteria (Table 1). These acceptance thresholds are those previously employed for the validation of other qPCR assays [17; 18; 19]. In summary, the linearity of the calibration curve was validated, with a high efficiency that ensures an optimal yield of PCR amplification. The method was considered repeatable and reproducible, and the limits of detection and quantification were established at 10 copies. The diagnostic performance was evaluated using 51 culture samples isolated from a diverse range of samples, including stool and environmental samples from laying hens, broilers, and breeding hens, all identified with the reference diagnostic method. All strains were correctly identified by the qPCR assay, showing optimal diagnostic specificity and sensitivity. In conclusion, the results of the parameters analysed met the acceptance criteria established for validation and were in agreement with the reference diagnostic method, making the assay suitable to distinguish the vaccine strain from wild _S. enterica_ subsp. _enterica_ sv. Typhimuriumium obtained from isolated colonies. The ability to accurately distinguish between vaccinated and infected animals ensures the integrity of surveillance programs and prevents unnecessary culling of vaccinated animals. The availability of this DIVA assay is essential for the safe use of the Primum Salmonella T vaccine in the field. To the best of our knowledge, this is the first available qPCR test for this purpose. DIVA assays based on qPCR, ELISA, or chromogenic media are available for other _Salmonella_ vaccines [20; 21; 22]. qPCR-based DIVA assays for other organisms have been validated with optimal diagnostic results [23; 24]. However, validations of _Salmonella_ qPCR DIVA assays have not been published, to the best of our knowledge. This product was registered in the Registry of Entities and Animal Health Products under the Ministry of Agriculture, Fisheries and Food of Spain (MAPA) with the following references: entity authorization number R-10046 and SalTypm&PriSal-T qPCR Duplex registration number 12186-RD. The SaITypm&PriSal-T qPCR Duplex assay has been conceived as an identification method for the analysis of _S. enterica_ subsp. _enterica_ sv. Typhimurium colonies obtained from pure cultures following ISO 6887-6:2013 and ISO 6579-1:2017 standards.