Multiplex PCR for Campylobacter Resistance

For all Campylobacter isolates, the presence of the following genes was individually assessed in each isolate: tet(O), tet(A), bla_OxA-61, the cluster of cmeA, cmeB, and cmeC, and the occurrence of Thr-86 to Ile mutations (C-to-T transition) in the quinolone resistance-determining region (QRDR) of the gyrA gene. The above established resistance to three different antibiotic classes: tetracycline, ampicillin, the CmeABC (efflux system) pump system, and quinolones, respectively. DNA extraction was carried out using the DNeasy^® UltraClean^® Microbial Kit (Qiagen Inc., Toronto, ON, Canada), according to the manufacturer’s instructions. The extracted DNA of each isolate was stored at −20 °C until further investigations, where it was then used as the template DNA in the PCR. The primer sequences and PCR conditions are listed in Table 2. The final amplification reaction volume was 20 µL, containing 2 µL of template DNA preparation from each Campylobacter isolate, 0.5 µL of each primer, 10 µL of (1X) QuantiNova SYBR Green PCR Master Mix (Qiagen, Hilden, Germany), and 7 µL of RNase-free water. The final reaction mixture volume was adjusted to 20 μL. Amplification was performed in a CFX96TM Real-Time PCR thermocycler (BioRad, CA, USA) with reaction conditions as suggested by Poudel et al. [36 (link)] and Hungaro et al. [35 (link)]. Simplex PCR assays for each gene were performed.