Prime safe dye

To identify related strains and distinguish between distinct strains, selected isolates of C. pseudotuberculosis were subjected to ERIC-PCR using the primer sequences ERIC1: 5´-ATGTAAGCTCCTGGGGATTCAC 3´ and ERIC2: 5´-AAGTAAGTGACTGGGGTGAGCG-3´ as described by Versalovic et al. (1991) (link). The ERIC-PCR was carried out in a total volume of 25 µl. Each reaction was made of 12 µl of hot start premix (Genedirex, Taiwan), 1 µl of each primer (10 pmol), 2 µl of sample DNA (30–100 ng/l), and 9 µl nuclease-free water (Qiagen, Germany). According to the PCR program used by Bakhshi et al. (2018) (link), the reaction was carried out using the 9700 GeneAmp PCR system (Applied Biosystems, USA) as follows: initial denaturation for 5 minutes at 94°C followed by 35 cycles of repeated steps of denaturation at 94°C for 1 minute, annealing at 54°C for 1 minute, extension at 72°C for 5 minutes, and final extension at 72°C for 10 minutes. On a 1% agarose gel with Prime Safe Dye (GeNet Bio, Korea) at 70 volts for 10 minutes and later at 80 volts for 1 hour, the PCR products were visualized. The GelJ gel analysis program (v. 2.0) was used to analyze the results of ERIC-PCR. The DICE similarity coefficient and a position tolerance of 1.0 were used in the unweighted pair group method with arithmetic averages (UPGMA) approach to create the dendrogram (Heras et al., 2015 ).