Synthesis of O-serogroup specific positive controls and real-time PCR standards for nine clinically relevant non-O157 STECs

► Controls from pathogens to evaluate the reliability of PCR assays may be difficult to obtain. ► We designed stacked synthetic real-time PCR standards to monitor clinically relevant STEC. ► We demonstrated advantages utilizing synthetic controls by (real-time) PCR assays. ► Synthetic controls were validated using DNA from native non-O157 STEC strains.