## 3 Results  
The SalTypm&PriSal-T qPCR Duplex kit, previously developed by GPS\({}^{\text{TM}}\) and commercially available, consists of a duplex assay for the simultaneous detection of _Salmonella enterica_ subsp. _enterica_ serovar Typhimurium and the Primun Salmonella T vaccine strain. The analytical specificity of the duplex assay was validated in vitro using genetic material from the Primun Salmonella T vaccine strain ST CAL16 Str+/Rif+/Enr-, _S. enterica_ subsp. _enterica_ sv. Typhimurium, _S. enterica_ subsp. _enterica_ sv. Enteritidis, _S. enterica_ subsp. _enterica_ sv. Typhi, _S. enterica_ subsp. _enterica_ sv. Paratyphi A, _S. enterica_ subsp. _arizonae_, and _S. enterica_ subsp. _salamae_. The internal control of the assay amplified in all samples, which indicates that there is no PCR inhibition (Figure 1). The PriSal-T assay gave positive results only for the Primun Salmonella T vaccine strain, while the SalTypm assay yielded positive results for both the Primun Salmonella T vaccine strain and the _S. enterica_ subsp. _enterica_ sv. Typhimurium reference strain (Figure 1). The other _S. enterica_ subspecies and serovars tested were negative for both assays. Therefore, the PriSal-T assay was specific for the Primun Salmonella T strain, and the SalTypm assay was inclusive for wild and vaccine strains of _S. enterica_ subsp. _enterica_ sv. Typhimuriumium and exclusive for the other _S. enterica_ subspecies and serovars tested. Several analytical sensitivity parameters of the duplex assay were evaluated in relation to established acceptance criteria. Each parameter was tested at least 10 times to ensure reproducibility. The study included qPCR standard curves generated using multiple template dilutions across six decimal ranges (from 10\({}^{6}\) copies to 10 copies). The template used for these assays is included in the kit as a positive control and can be used to generate a standard curve from 10\({}^{4}\) to 10 copies. For each individual assay, the slope (a) and coefficient of determination (R\({}^{2}\)) were calculated for the mean of 15 standard curves to assess the linear regression. The SalTypm and PriSal-T assays had slopes of \(-\)3.322 and \(-\)3.321, respectively, and both achieved an R\({}^{2}\) value of 1.000, meeting the criteria for acceptability. A Fisher test with a 95% confidence interval and \(n=10\) was performed to validate the linear model. The F\({}_{\text{fisher}}\) value was 5.318 for both assays, and the F\({}_{\text{assay}}\) values were 0.572 and 0.145 for the SalTypm and PriSal-T assays, respectively. Since the F\({}_{\text{assay}}\) values were lower than the F\({}_{\text{fisher}}\) value, the linear model was validated for both assays. The efficiency was 100.0% for the mean of 15 standard curves for both assays, falling within the acceptability range (75-125%). Repeatability is the ability of a test to produce the same results under the same conditions, while reproducibility is the ability of a measurement or test to produce consistent results under different conditions. Standard curves were performed by the same technician with one template decimal dilution to evaluate the repeatability of the assays and by two technicians with different template decimal dilutions to evaluate reproducibility. The coefficient of variation (CV) of the Ct values ranged from 0.37% to 1.39% for repeatability, and from 0.92% to 2.43% for reproducibility in the SalTypm assay and from 0.28% to 1.30% for repeatability, and from 1.08% to 1.83% for reproducibility in the PriSal-T assay. All CV values were below 10% and therefore the repeatability and the reproducibility results were acceptable. Both assays were thus repeatable and reproducible. The limit of detection (LOD) and the limit of quantification (LOQ) are the lowest number of copies that can be detected and quantified. In these experiments, the lowest number of copies analysed was 10 copies.