2.5. Detection of Campylobacter and ARGs using standard Polymerase Chain Reaction (PCR)

The primers C412F and C1228R were used to detect the Campylobacter 16S rRNA [21]. Detection of hippuricase-positive Campylobacter was done by targeting the hipO gene [22]. Tetracycline-resistant genes were detected using the tetO primers while the primers cmeA, cmeB, and cmeC were used to detect, Campylobacter multi-drug resistant genes. The primers used for cmeA, cmeB, cmeC, and tetO are specific for Campylobacter jejuni [23,24]. Resistance to erythromycin resistance was determined by detecting the A2074G and A2075G point mutations of Campylobacter on the 23s rRNA gene. The primers used in this study were manufactured by Inqaba Biotech, South Africa. The PCR was carried out using the primer sets and optimised protocols as described in Table 1, Table 2 respectively. The PCR reaction mixture was 50 μL and consisted of the following; 25 μL EmeraldAmp GT PCR master mix (Takara Bio Inc, China), 2 μL forward, 2 μL reverse primers, 2 μL template DNA, and 19 μL molecular grade water. The amplicons were visualised by electrophoresis on 1.5% agarose gels (CSL-AG100, Cleaver Scientific Ltd. Warwickshire, UK) and the gel images were captured using a UV machine (molecular imager ChemiDocTM XRS+, BIO-RAD).

Primers used to detect Campylobacter and antibiotic-resistant genes.

Primers and the PCR amplification conditions for the genes detected in the study.