Two typing methods (ERIC-PCR and BOX-PCR) were used to genotype the P. aeruginosa clinical isolates, in order to study the bacterial genetic diversity within this complex group. The ERIC and BOX primer sequences (Macrogen, South Korea) were used in PCR to detect differences in the number and distribution of these repetitive sequences in the bacterial genome (Table 1 ) (14 (link), 15 (link)).
PCR amplification was performed in a final volume of 25 μL containing 3 μL of purified total DNA, 8 μL of PCR master mix (containing Taq polymerase, MgC12, dNTPs, and PCR buffer; Amplicon, Denmark), and 2.5 pM of each primer (Macrogen, South Korea). PCR was carried out in a thermal cycler apparatus (PeqStar; PeqLab, Germany) with an initial denaturation step at 95°C for 3 minutes, followed by 35 cycles including denaturation at 94°C for 1 minute, annealing (at 52°C for ERIC-PCR and at 48°C for BOX-PCR) for 1 minute and extension at 72°C for 2 minutes, with a final extension step at 72°C for 5 minutes.
PCR amplification was performed in a final volume of 25 μL containing 3 μL of purified total DNA, 8 μL of PCR master mix (containing Taq polymerase, MgC12, dNTPs, and PCR buffer; Amplicon, Denmark), and 2.5 pM of each primer (Macrogen, South Korea). PCR was carried out in a thermal cycler apparatus (PeqStar; PeqLab, Germany) with an initial denaturation step at 95°C for 3 minutes, followed by 35 cycles including denaturation at 94°C for 1 minute, annealing (at 52°C for ERIC-PCR and at 48°C for BOX-PCR) for 1 minute and extension at 72°C for 2 minutes, with a final extension step at 72°C for 5 minutes.
