Multiplex PCR for Shiga Toxin E. coli

Shiga toxins 1 and 2, O157 (RfbE), and H7 (Flich7) target genes were detected in isolated E. coli strains using multiplex PCR. The PCR reaction was prepared in 20 μL final volume which contained 8.2 μL of nuclease-free water, 5 μL of the DNA template, 4 μL of ready 5× Hot FIREPol® Blend Master Mix Ready to load [Hot FIREPol® DNA Polymerase, proofreading enzyme, 5× Blend Master Mix Buffer, 12.5 mM Mgcl₂, 2 mM dNTPs (Solis Biodyne, Estonia)], and selected primers (Table-1) to a final concentration of 10 μM [7 (link),15 (link)]. The thermocycling conditions were set at initial denaturation of 95°C for 15 min, followed by 25 cycles of 94°C for 30 s, 65°C for 90 s, 72°C for 90 s, and final extension at 72°C for 7 min. Amplified samples were evaluated by 1.2% agarose gel electrophoresis in 1× of TBE buffer, ethidium bromide (0.5 μg/mL) staining and visualized under UV illumination [16 (link)].