Genus-specific dsbA gene amplification

PCR was performed in a Bio-Rad T100™ Thermal cycler (Bio-Rad, Hercules, CA, USA) according to the guidelines described by Iweriebor et al. [9 (link)] for amplification of the genus-specific disulfide bond formation protein (dsbA) gene using the following universal primers: EHL dsb F 5′-GAT GAT GTC TGA AGA TAT GAA ACA AAT-3′ and EHL dsb R 5′-CTG CTC GTC TAT TTT ACT TCT TAA AGT-3′ to generate 409 bp fragments. PCR was performed in a 25 μL reaction mixture containing 12.5 μL of One Taq^® quick-load^® master mix (New England BioLabs, Ipswich, MA, USA), 1 μL of 10 pMol for each of the forward and reverse primers, 8.5 μL of nuclease-free water, and 2 μL of DNA template. The cycling conditions were as follows: an initial heating block at 94 °C for 3 min, followed by 35 cycles of denaturation at 93 °C for 30 s, then annealing at 47 °C for 30 s, with an elongation at 72 °C for 1 min and a final elongation at 72 °C for 5 min. Nuclease-free water was used as a non-DNA negative control and positive control was E. ruminantium DNA obtained from a collaborator at the Center for Zoonosis Control, Hokkaido University, Japan. PCR products were visualized by electrophoresis on a 1% agarose gel stained with ethidium bromide at 100 volts for 45 min. Positive PCR products were sent to Inqaba Biotech (Pty), Ltd., Pretoria (South Africa) for purification and sequencing.