Preparation of milk samples for PCR analysis using a rapid filtration technique.
OBJECTIVE
To investigate the usefulness of a straightforward filtration method for the isolation of Escherichia coli O157:H7 contaminants from milk for PCR detection.


RESULTS
Escherichia coli O157:H7 is grown in milk and enriched in Luria-Bertani (LB) medium. Samples are filtered through a 0.45-microm pore membrane. The membrane is immersed in 200-microl lysis buffer and incubated at 95 degrees 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.


CONCLUSIONS
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 degrees C for 10 min.


CONCLUSIONS
The simplicity of this method allows inexpensive, high throughput automation that meets the demands of modern food hygiene monitoring.
Wu
S-J
SJ
Department of Life Sciences, National Central University, Jung-da Road, Jhong-li City, Taiwan. jyewu@cc.ncu.edu.tw
Kado
C I
CI
D000818:Animals; D004269:DNA, Bacterial / Q000302:isolation & purification; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005506:Food Contamination / Q000517:prevention & control; D005516:Food Microbiology; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D017751:Safety Management / Q000379:methods; D014462:Ultrafiltration / Q000379:methods
D004269:DNA, Bacterial