Disclosed are methods and kits for the specific detection of E. coli O157:H7 and not E. coli O55:H7 from samples such as: complex food matrices, water, beverages, fermentation broths, forensic & biological samples, and environmental samples including food processing and manufacturing surfaces. In some embodiments, a method of the disclosure comprises: hybridizing at least a first pair of polynucleotide primers to at least a first target polynucleotide sequence, hybridizing at least a second pair of polynucleotide primers to at least a second target polynucleotide sequence, amplifying the at least first and at least second target polynucleotide sequences, and detecting the first and second amplified target polynucleotide sequence products, wherein the detection of both the first amplified target polynucleotide sequence product and the second amplified target polynucleotide sequence product is indicative of the presence of E. coli O157:H7 in a sample and not E. coli O55:H7.