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Preparation of milk samples for PCR analysis using a rapid filtration technique
Author(s) -
Wu S.J.,
Kado C.I.
Publication year - 2004
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2004.02259.x
Subject(s) - filtration (mathematics) , chromatography , escherichia coli , bacteria , contamination , lysis , dna extraction , microbiology and biotechnology , membrane filter , chemistry , biology , lysis buffer , membrane , food science , polymerase chain reaction , biochemistry , gene , ecology , statistics , genetics , mathematics
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‐ μ m pore membrane. The membrane is immersed in 200‐ μ l 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.