## 4 Discussion  
The comprehensive results obtained from the validation of the SalTypm&PriSal-T qPCR Duplex kit demonstrate its high specificity, sensitivity, and reliability in distinguishing between the vaccine and wild strains. These results provide a strong basis for discussing the \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & \multicolumn{3}{c}{**SalTypm**} & \multicolumn{2}{c}{**PriSal-T**} \\ \hline  & **Parameter** & \multicolumn{2}{c}{**Obtained Value**} & \multicolumn{2}{c}{**Acceptance Criteria**} & **Result** \\ \hline \multirow{4}{*}{**Standard curve** **(\(\mathbf{\textit{tr}=10}\))**} & Estimation of the & Y = \(-\)3.322*X + 39.243 & Y = \(-\)3.321*X + 38.128 & \multirow{4}{*}{**Accepted**} \\  & regression line & a = \(-\)3.322 & a = \(-\)3.321 & \(-\)4.115 \(<\) a \(<\) \(-\)2.839 & \\  & Validation of the & F\({}_{\text{easy}}\) = 0.572 & F\({}_{\text{easy}}\) = 0.145 & & \multirow{4}{*}{\(\text{F}_{\text{\emph{singary}}}\) \(<\) F\({}_{\text{\emph{Fisher}}}\)} & Regression model \\  & linear model & F\({}_{\text{\emph{Fisher}}}\) = 5.318 & F\({}_{\text{\emph{Fisher}}}\) = 5.318 & & & **Accepted** \\  & Validation of & Efficiency (\(\epsilon\)) = 100.0\% & Efficiency (\(\epsilon\)) = 100.0\% & 75\% \(<\) \(\epsilon\) \(<\) 125\% & \\  & & Repeatability & & & & **Validated** \\  & Concentration & CV (\%) & CV (\%) & & & \\  & \(10^{6}\) copies & 0.37 & 0.35 & & & \\  & \(10^{5}\) copies & 0.42 & 0.44 & & & \\  & \(10^{4}\) copies & 0.42 & 0.36 & CV \(<\) 10\% & **Repeatable** \\  & \(10^{3}\) copies & 0.40 & 0.28 & & & \\  & \(10^{2}\) copies & 0.66 & 0.34 & & & \\  & \(10^{1}\) copies & 1.39 & 1.30 & & & \\ \cline{2-5}  & Reproducibility & & & & & \\  & Concentration & CV (\%) & CV (\%) & & & \\  & \(10^{6}\) copies & 1.35 & 1.61 & & & \\  & \(10^{5}\) copies & 1.42 & 1.77 & & & \\  & \(10^{1}\) copies & 1.14 & 1.18 & CV \(<\) 10\% & **Reproducible** \\  & \(10^{3}\) copies & 1.03 & 1.12 & & & \\  & \(10^{2}\) copies & 0.92 & 1.08 & & & \\  & \(10^{3}\) copies & 2.43 & 1.83 & & & \\ \hline \multirow{2}{*}{**Detection limit**} & \multirow{2}{*}{10 copies} & \(\mathbf{n=15}\) & \(\mathbf{n=15}\) & \(\mathbf{n=15}\) & \multirow{2}{*}{Posit. \(\geq\) 90\%} & **Accepted** \\  & & Post. = 100\% & Posit. = 100\% & & & \\ \hline \multirow{2}{*}{**Quantif. limit**} & \multirow{2}{*}{10 copies} & \(T_{\text{\emph{Fisher}}}\) = 1.500 & \(T_{\text{\emph{Fisher}}}\) = 0.581 & \multirow{2}{*}{\(T_{\text{\emph{Fisher}}}\) \(<\) \(T_{\text{\emph{Fisher}}}\)} & **Accepted** \\  & & \(T_{\text{\emph{Fisher}}}\) = 2.145 & \(T_{\text{\emph{Fisher}}}\) = 2.145 & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Obtained values, criteria for acceptance, and results of the SalTypm&PriSal-T qPCR Duplex kit (\(\mathbf{\textit{tr}=\text{number of replicates for each parameter}}\)). \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**SalTypm\&PriSal-T qPCR Duplex Kit** & \multicolumn{3}{c}{**Reference Assay (Antibiogram Testing)**} \\ \hline
**Number of samples** & **51** & **Positive samples** & **10** & **Negative samples** & **41** \\ Positive samples for PriSal-T & 10 & True Positives & 10 & False Positives & **0** \\ Negative samples for PriSal-T & 41 & False Negatives & 0 & True Negatives & 41 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Diagnostic specificity and sensitivity results for the SalTypm&PriSal-T qPCR Duplex kit. implications of using this assay in the effective management of _Salmonella_ infections within poultry production systems. The accurate differentiation between vaccinated and infected animals is crucial for ensuring the success of vaccination programs while maintaining robust surveillance and outbreak prevention strategies. Bacteria of the genus _Salmonella_ spp. are one of the most relevant foodborne pathogens worldwide, with _S. enterica_ subsp. _ enterica_ sv. Typhimurium being one of the predominant serotypes associated with human infections. Consumption of poultry products is the primary source of transmission to humans. The use of live attenuated _S.