Agarose gel electrophoresis

The amplification of the 16S rRNA was performed as described by Moyaert et al. (2008) (link). The total volume of 25 μl consisted of 5 μl of deoxynucleoside triphosphate mix, 2.5 μl of 10 × PCR buffer, 0.25 μl of the primer, and 1 μl of the DNA template. The amplification of the ureC gene was performed according to the method described by Kianpour et al. (2014) . The total volume of 50 µl consisted of 5 µl 10 × buffer + MgCl2, 2 mM dNTP, 2 unit Taq DNA polymerase, 100 ng DNA template, and 25 pmol (pmol) of each primer. The amplification of the babA2, cagA and vacA virulence genes was performed as previously described by Paniagua et al. (2009) . In brief, a total volume of 25 μl (2.5 pmol of babA2-F and babA2-R, 25 pmol of vacA-F and vacA -R, 10 pmol of cag5c-F and cag3c-R, 0.25 mM of each dNTP, 0.9 U of Taq DNA polymerase and 1.5 mM of MgCl2) was applied. Taq polymerase, MgCl₂, and nuclease-free water were utilized appropriately in each test. The amplification of all genes was performed using a Thermal Cycler (Master cycler, Eppendorf, Hamburg, Germany). The amplifications were performed as shown in Table 1. Finally, the amplified DNA fragments of all genes were investigated using 2% agarose gel electrophoresis (AppliChem, Germany, GmbH) in 5 μl/100 ml Tris-borate-EDTA (TBE) buffer stained with ethidium bromide and visualized using an ultra-violet (UV) transilluminator.

Genomic DNA was isolated using the Gene JET Genomic DNA Purification Kit (Fermentas), as reported by Sambrook et al. (1989 ). The purified DNA was immediately used in the downstream application or kept at −20°C.
Multiplex PCR was used to identify Shiga toxins (stx1 and stx2), intimin (eaeA), and haemolysin (hylA) in E. coli using specific primers (Pharmacia Biotech) and cycling conditions for stx1 and stx2 according to Dhanashree and Mallya (2008 (link)), eaeA according to Mazaheri et al. (2014 ), and hylaA according to Fratamico et al. (1995 (link)). The amplified DNA fragments were analyzed using 2% agarose gel electrophoresis (Applichem, Germany, GmbH) in 1× TBE buffer, stained with ethedium bromide, collected, and seen under a UV transilluminator. The fragment size was evaluated with a 100-bp plus DNA ladder (Qiagen, Germany GmbH).