(vii) qPCR primer development and assay conditions.

For each bacterial strain used to inoculate milk samples, qPCR primers were designed to target the RNA polymerase subunit beta gene (rpoB) because it is a single-copy gene and allows for a more accurate comparison between bacterial numbers than 16S rRNA genes. Primers were also designed to target a conserved region of the 16S rRNA gene, and calculated 16S copy numbers were used as a proxy for total bacterial numbers. Primer details are described in Table 7. Reactions were carried out in duplicate using 2 μl of extracted DNA. The final qPCR volumes totaled 25 μl and contained 12.5 μl SYBR green master mix (ThermoFisher Scientific), 2 μl extracted DNA, 0.5 μM forward primer, 0.5 μM reverse primer, and 9.5 μl nuclease-free water. Reactions were carried out in a QuantStudio 6 instrument (ThermoFisher Scientific), with the following cycling conditions: 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min and a melting curve of 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s.

Primers used in this study

Bovine genome copy numbers were calculated using a commercial TaqMan assay targeting the UXT gene (ThermoFisher Scientific). The final qPCR totaled 20 μl and included 1 μl of the gene expression assay mixture, 10 μl TaqMan Fast advanced master mix (ThermoFisher Scientific), 7 μl of nuclease-free water, and 2 μl of the template. Reactions were carried out in a QuantStudio 6 instrument, with the following cycling conditions: 95°C for 20 s, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. For each of the three biological replicates, samples extracted with all kits were amplified in a single PCR plate and compared using the same standard curve.