Two 5’nuclease‐based PCR methods (PCR‐LS‐50B and PCR‐7200) were evaluated to determine their sensitivity for detecting Escherichia coli O157:H7 from pure cultures and in food samples enriched in different media and after different incubation periods. The PCR‐7200 method was able to detect E. coli O157:H7 at ± 102 CFU/mL in pure culture in both mECB and EEB. In spiked meat samples, the PCR‐7200 procedure was capable of detecting the eaeA gene at lower concentrations than the PCR‐LS‐50B procedure, regardless of the meat type or enrichment medium. Escherichia coli O157:H7 spiked at 0.3 CFU/mL was detectable after 9 h in EEB, but it was not detected in mECB within 24 h. An enrichment time of 4 h in mECB was needed to detect E. coli O157:H7 when spiked at higher levels (41 CFU/mL). The detection levels reported in this study are similar with other reported PCR‐based detection techniques for E. coli O157:H7, however, the 5’nuclease‐based assays are less labor intensive and capable of higher sample throughput because of their automated detection and analysis steps.