PCR amplification of virulence-related genes of P. multocida as well as antimicrobial resistance genes such as erm(X) (macrolide, lincosamides and streptogramins resistance), sul1(sulfonamide resistance), tet(H) (tetracycline resistance), bla ROB-1 (beta-lactam resistance), mcr1(colistin resistance), and dfrA1 (trimethoprim resistance) were performed by PCR assays using the oligonucleotide primer sequences presented in Table
A PTC-100 programmable thermal cycler (Peltier-Effect cycling, MJ, UK) was used to conduct the PCR assays. The reaction mixture’s final volume was adjusted to 25 μL and comprised 12.5 μL of DreamTaq TM Green Master Mix (2X) (Fermentas, USA), 0.4 μL of each primer at 100 pmoL (Sigma, USA), 5 μL of template DNA, and 25 μL of nuclease-free water. The cycling conditions were: 30 cycles; 95 °C for 45 s, primer annealing (TA, Table
On a 1.5% agarose gel (Applichem, Germany, GmbH), the PCR products were separated by electrophoresis, and the gel was photographed using a gel documentation framework (Alpha Innotech, Biometra). The data was analyzed by computer software. As a quality control, P. multocida ATCC® 43137™ was utilized.