Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) for E. coli isolates

ERIC-PCR was performed on a total of 37 E. coli isolates with primers ERIC-1 (5′-ATG TAA GCT CCT GGG GAT TCA C-3′) and ERIC-2 (5′-AAG TAA GTG ACT GGG GTG AGC G-3′) that were described in previous studies²⁰. Genomic DNA was extracted from 1 mL of overnight culture using a G-spin™ Genomic DNA Extraction Kit (iNtRON, Korea) and following the manufacturer’s instructions. ERIC-PCR was performed in a total volume of 20 μL containing 0.4 μM concentrations of each forward and reverse primer, 20 ng of DNA template and 1× Green PCR master mix kit (Biotechrabbit, Germany). The amplification steps were completed using a BiometraTOne96G thermocycler (AnalytikJena, Germany) with the following thermal cycles: the initial denaturation at 94 °C for 5 min, followed by 35 cycles (denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, and extension at 72 °C for 5 min), and a final extension step at 72 °C for 10 min. PCR products were separated using 2.0% agarose gel electrophoresis, stained with 1X GelRed (Sigma Aldrich, USA), and visualized under a UV transilluminator UVP GelStudio (AnalytikJena, USA). ERIC-PCR results were analyzed by online data analysis services (insilico.ehu.es). ERIC profiles were compared using the Dice coefficient method, and a dendrogram was made via the unweighted pair-group method using arithmetic averages (UPGMA).