Enterohemorrhagic Escherichia coli (EHEC) is a gramnegative bacterium and an intestinal pathogen that can cause life-threatening complications such as hemolytic uremic syndrome (HUS). A major virulence factor for EHEC with the ability to cause disease is the production of Shigatoxin 1 and 2 (Stx1 and Stx2). In recent years, more subtypes of Stx1 and Stx2 that can cause severe diseases have been reported. The purpose of this study was to create a new In-house PCR protocol for verifying EHEC colonies, with the presence of stx1 and stx2. This study included the reference strain E. coli D2154 (positive for stx1 and stx2) and a strain positive for stx2f. Optimization of the protocol was performed by both conventional and real-time PCR. When designing multiplex real-time PCR, the probe concentration 0,1 µM with a primer concentration of 0,3 µM for stx1 and the probe concentration 0,2 µM with a primer concentration of 0,5 µM for stx2 was used. The new protocol was validated by testing saved material from 42 faecal samples, which had previously been evaluated both in the laboratory and at a reference laboratory. The efficiency of the new protocol was calculated to be 91 % for stx1 and 95 % for stx2. The new protocol had a specificity between 56 – 100 % and a sensitivity between 88 – 100 % in comparison to the reference laboratory. The new protocol was able to detect stx2f in samples that were negative at the reference laboratory, which was the reason for many false positive results and low specificity. The ability of the new protocol to detect stx1 and stx2 in EHEC colonies was convincing, with a sensitivity and specificity of 100%. The same applies to the protocol’s ability to detect the subtype stx2f. Thus, the new protocol will eventually, after further optimization, be implemented and replace the existing protocol at Clinical Microbiology in Lund.