Preparation of milk samples for PCR analysis using a rapid filtration technique

Aims: To investigate the usefulness of a straightforward filtration method for the isolation of Escherichia coli O157:H7 contaminants from milk for PCR detection. Methods and Results: Escherichia coli O157:H7 is grown in milk and enriched in Luria-Bertani (LB) medium. Samples are filtered through a 0.45-μm pore membrane. The membrane is immersed in 200-μl lysis buffer and incubated at 95°C for 10 min to release bacterial DNA for subsequent PCR detection. Under current conditions, the overall duration from filtration to PCR-ready DNA generation is <20 min, and the detection level for PCR was as low as 10 CFU of bacteria in 1 ml of milk. Conclusion: Bacterial contaminants of milk can be concentrated and isolated by a simple, one-step filtration and their DNA can be released for subsequent PCR detection by heating the filter membrane at 95°C for 10 min. Significance and Impact of the Study: The simplicity of this method allows inexpensive, high throughput automation that meets the demands of modern food hygiene monitoring.