Corbett cas1200 robot

The qPCR was performed with the SensiFAST SYBR Hi-ROX Kit (Bioline, Australia) following the manufacturer’s protocol. The master mix was prepared and applied to reaction wells through Corbett CAS1200 robot (Corbett Life Science, Australia). The final reaction volume of 20 μL contained 10 μL SensiFAST SYBR Hi-Rox mix, 1 μL of each of the forward and reverse primers (10 μM concentration), 6 μL RNase-free water and 2 μL of DNA template. The reaction was performed in duplicates in a Rotor-Gene Disc 100 (Qiagen, Australia) with a Rotor-Gene 6000 Thermocycler (Corbett Life Science, Australia). Each run contained no-template control for ruling out external contaminations. The three-step cycling conditions included an initial denaturation at 95°C for 3 min, then 40 cycles of denaturation at 95°C for 5 s and annealing and extension at 59°C (Table 1) and 72°C for 20 and 30 s, respectively. During qPCR cycles, the fluorescence detection was conducted at the end of each annealing step, and a melting curve analysis step (at a ramp from 55 to 95°C) was included to assess the specificity of the amplification.

The extracted DNA samples were subjected to quantitative PCR for Salmonella Typhimurium quantification from week 1 post infection until sacrifice. The qPCR was performed using SensiFAST SYBER HI-ROX Kit (Bioline, Australia) following the manufacture’s protocol. The qPCR reaction was performed in 72 well rotor-gene disc (Qiagen, Australia). The master mix was prepared and added to the disc through Corbett CAS1200 robot (Corbett Life Science, Australia). The 10 μL final reaction volume contained 5 μL SensiFAST SYBER Hi-Rox mix, 1 μL each of the forward (5′-TTTACCTCAATGGCGGAACC) and reverse (5′-CCCAAAAGCTGGGTTAGCAA) TSR3 primers, 1 μL RNase-free water and 2 μL DNA template. The cycling conditions were initial denaturation at 95 °C for 3 min, then 40 cycles of denaturation at 95 °C for 5 s, annealing and extension at 59 °C and 72 °C for 20 and 30 s, respectively.
A standard curve was constructed by spiking non-infected control faeces with known amount of Salmonella Typhimurium. Briefly, serial ten-fold dilutions of Salmonella Typhimurium grown overnight in LB broth were used to spike the faecal samples. The DNA was extracted from the spiked faecal samples to construct a standard curve and limit of detection was established.