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Aims: The purpose of this study was to develop a reliable molecular procedure for the detection of Escherichia coli in milk.

Methods and Results: Robust and expeditious DNA extraction and PCR techniques were evaluated using Enzyme-Linked Immunosorbent Assay (ELISA) detection of biotin-labelled amplicons to facilitate optimal detection of E. coli DNA.

Conclusions: It was found that 5 E. coli colony-forming units (cfu) could be detected per PCR reaction using the PCR-ELISA system, equating to a sensitivity of detection of 100 E. coli cfu ml−1 pasteurized milk.

Significance and Impact of the Study: This approach should facilitate evaluation of milk contamination and enable rapid detection of E. coli mastitis, leading to correct deployment of relevant antibiotic therapy and improved animal welfare.