Biotyping Pasteurella multocida via RAPD-PCR

RAPD-PCR was performed using the extracted DNA to investigate the biotype diversity of P. multocida in the study isolates [33 (link),34 ]. The RAPD-PCR was performed using (5´-GCGATCCCCA-3´) primer following the previously optimized protocol for Escherichia coli [22 ]. Using previously published pathogenic gene-specific primers (Table-1) for PCR assays, we surveyed ten pathogenic genes in the strains of P. multocida [9 (link),16 (link)]. Briefly, the PCR mixer possessed 2 mL DNA template (300 ng/mL), 10 mL PCR master mix 2X (GoTaq^® Colorless Master Mix, Promega, Madison, WI 53711 USA) and 1 mL (100 pmol/mL) of each primer (Table-1) in each reaction tube. The cycling condition for PCR amplifications was as follows: 94°C for 5 min; 35 cycles of 1min at 94°C, 1 min at 50-60°C, and 1 min at 72°C; and 72°C for 7 min. Amplified PCR products were visualized on 1.5% agarose gel prepared in 1× TAE buffer. After gel electrophoresis, the images were captured using Image ChemiDoc™ Imaging System (Bio-Rad, USA) [9 (link)].