Sample preparation for PCR detection of Enterohaemorrhagic E.coli in food

  Objective   To develop a rapid, sensitive and cost-effective sample preparation for PCR detection of EHEC O157:H7 in ground beef.

  Methods   Ground beef samples were inoculated with varying concentrations of E.coli O157:H7.PCR inhibitors were removed and bacterial cells were concentrated by filtration and centrifugation.DNA was purified and concentrated using enzymatic digestion and successive freeze/thaw cycles and the Shiga toxin gene was amplified using PCR to evaluate the sample preparation method.

  Results   Without prior enrichment of cells in broth media, the detection limit was 10 ³ CFU/g beef.When a 6 h enrichment step was incorporated, the detection limit was 1 CFU/g beef.The total time required from beginning to end of the procedure was 12 h.

  Conclusion   The sample preparation method can substantially improved the sensitivity of E.coli O157:H7 by PCR detection.