Invitrogen 1 kb plus ladder

ERIC-PCR was performed for molecular typing using the primer ERIC2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′). 2 μL of DNA template were mixed with 12.5 μL of DreamTaq^TM Green PCR Master mix (Thermo Scientific, Vilnius, Lithuania), 1 μL of primer (10 pmol), and an adequate volume of sterile nuclease-free water to make a 25 μL reaction mixture. The PCR was carried out in a Bio-Rad T100™ Thermal Cycler (Bio-Rad, Hercules, CA, USA), with the following protocol: initial denaturation at 94 °C for 15 min, followed by 40 cycles of amplification. Denaturation at 95 °C for 1 min, primer annealing at 37 °C for 1 min, and primer extension at 72 °C for 1 min were used in each cycle. After the amplification cycles, samples were kept at 72 °C for 10 min to allow partially produced DNA to extend [78 (link)]. Then, 2% w/v agarose gel electrophoresis was used to visualize ERIC fragments, and the data were evaluated using GelJ v.2.0 software (Open source Java software) [83 (link),85 (link)]. Invitrogen 1 kb plus ladder (Thermo Fisher Scientific, Waltham, MA, USA) was used to normalize the fragment patterns in GelJ v.2.0 software [27 (link)].

For the investigation of genetic relatedness among the pathogenic isolates, Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences PCR was performed using ERIC2 primer (5′-AAGTAAGTGACTGGGGTGAGCG-3′). The PCR was carried out as per the protocol mentioned before (54 (link)). The amplified PCR products were resolved on 2% agarose gel. Gels were run at 90 volts for ~3 h. Invitrogen 1 kb plus ladder (Thermo Fisher Scientific, US) was used in the very first and last lanes per gel. GelJ v.2.0 software was used for gel image analysis (55 (link)). The gaussian regression method was used to normalize the image. Clusters of ERIC-PCR patterns were generated through dice coefficient and the unweighted pair group method using arithmetic averages (UPGMA) with 1.0% tolerance value.