In this study number of 130 bulk tank milk samples which were delivered to Pegah pasturisation factory in Mashhad were collected randomly, during the summer months. For isolation of Escherichia coli O157:H7, samples were firstly enriched in modified trypticase soy broth containing novobiocin, followed by plating onto sorbitol Mac Cankey agar supplemented with cifixime and potassium tellurite. Consequently the suspected non sorbitol fermenting(NSF) colonies were confirmed by biochemical tests as Escherichia coli and then employed for multiplex-PCR assay, using primers specific for O157 and H7 antigens gene and then primers specific for stx1 and stx2 gene. In this study NSF Escherichia coli colonies were recovered from 8 samples, and in multiplex-PCR assay one sample (0.77%) confirmed as Escherichia coli O157:H7. The second multiplex PCR assay showed that the isolate were harboring the stx2 gene. The PCR assay employed in this study may be a possible alternative to immunological assays which detects somatic and flagellar antigens, besides this procidure determines the potential of isolates for toxin production .

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