## 2 Materials and Methods
### Real-Time PCR for the Amplification of Seven Bacterial Pathogens in the Milk Samples  
An RT-PCR assay was developed in order to qualitatively identify and successfully differentiate _Shigella_ spp. _E. coli, Salmonella_ spp., _Campylobacter_ spp., _Adenovirus F_, _Rotavirus A_, _No-rovirus 2 genotype_, and _Astrovirus_, as per the Saccace Biotechnology Kit (Acute Intestinal Infection-A.I.I.-Screen Real-TM) instructions. Four reaction mixes had to be prepared in new sterile tubes for each one of the samples (PCR-mix-1 _Shigella_ spp./_Salmonella_ spp., PCR-mix-1 _Campylobacter_ spp./_Adenovirus_, PCR-mix-1 _Rotavirus/Astrovirus_, and PCR-mix-1 _Norovirus_/IC). The interpretation of the results is achieved (software included in the device) when the fluorescence curve crosses the threshold line as follows: PCR-mix-1 Norovirus/IC is used to detect the internal control (IC) on the (Green) FAM channel along with Norovirus on the (Yellow) JOE channel. On the same channels, Rotavirus A and Astrovirus are detected when PCR-mix-1 Rotavirus/Astrovirus is used, respectively. On the FAM channel _Shigella_ spp. summer. _E. coli_ was also detected with PCR-mix-1 _Shigella_ spp./_Salmonella_ spp., while _Salmonella_ spp. was detected on the JOE channel. Finally, with PCR-mix-1 _Campylobacter_ spp./ _Adenovirus_ on the FAM channel, there was the detection of _Campylobacter_ spp., while _Adenovirus_ was detected on the JOE channel. A Ct value less than 40 is indicative of a positive sample.  
_Analytical specificity:_ The negative samples validated the analytical specificity of the primers and probes, as they did not generate any signal with the above-mentioned tested microorganisms. The A.I.I. kit's specificity was 100%.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Target Genes** & **Primer Sequence** & **Amplicon Size (bp)** \\ \hline _Enterococcus_ spp. & 5\({}^{\prime}\)-TACTGACAAACCATTCATG-3\({}^{\prime}\)R & 112 \\  & 5\({}^{\prime}\)-AACTTGCTCCCAACCGGAAC-3\({}^{\prime}\)F & \\ _Staphylococcus epidermidis_ & 5\({}^{\prime}\)AGTACAGAACCGTATTGCCTGGCT3\({}^{\prime}\)R & 720 \\  & 5\({}^{\prime}\)TGAAGTCAATTCGGTCTCGCGT3\({}^{\prime}\)F & \\ _Staphylococcus saprophyticus_ & 5\({}^{\prime}\) TCAAAAGTTTTAAAAAATTAC3\({}^{\prime}\)R & 221 \\  & 5\({}^{\prime}\) GGGCGTCCACAAAATCAAGGAGG\({}^{\prime}\)F & \\ _Klebsiella pneumoniae_ & 5\({}^{\prime}\)-TTGAAAGGTTTCGAAACGT-3\({}^{\prime}\)R & 130 \\  & 5\({}^{\prime}\)-TTCACTTGAAGTTTTCTGTTC-3\({}^{\prime}\)F & \\ _E. coli_ & 5\({}^{\prime}\)-GAC CTC GGT TTA GTT CAC A-3\({}^{\prime}\)R & 585 \\  & 5\({}^{\prime}\)-CAC ACG CTG ACG CTG ACC A-3\({}^{\prime}\)F & \\ _Staphylococcus aureus_ & 5\({}^{\prime}\)-TCG CTT GCT ATG ATT GTG G-3\({}^{\prime}\)R & 359 \\  & 5\({}^{\prime}\)-GCC AAT GTT CTA CCA TAG C3\({}^{\prime}\)F & \\ \hline \hline \end{tabular}
\end{table}
Table 1: Primer sequences used for the detection of pathogens related to mastitis using conventional PCR.  
Analytical sensitivity:_ With the A.I.I., the specific microorganisms can be detected in all tests (100%) with a minimum sensitivity of 500-1000 copies/mL. Detection was performed using the control standard while dilutions were used for the negative samples.