US Department of Agriculture, Food Safety and Inspection Service, Washington D.C., USA.
Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef
Article first published online: 16 JUN 2003
Journal of Applied Microbiology
Volume 95, Issue 1, pages 129–134, July 2003
How to Cite
Cui, S., Schroeder, C.M., Zhang, D.Y. and Meng, J. (2003), Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef. Journal of Applied Microbiology, 95: 129–134. doi: 10.1046/j.1365-2672.2003.01951.x
- Issue published online: 16 JUN 2003
- Article first published online: 16 JUN 2003
- 2002/378: received 15 October 2002, revised 25 February 2003 and accepted 5 March 2003
- E. coli O157:H7;
- ground beef;
Aim: To develop an improved, rapid and sensitive sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef.
Methods and Results: Fresh ground beef samples were experimentally inoculated with varying concentrations of E. coli O157:H7. PCR inhibitors were removed and bacterial cells were concentrated by filtration and centrifugation, and lysed using enzymatic digestion and successive freeze/thaw cycles. DNA was purified and concentrated via phenol/chloroform extraction and the Shiga toxin 1 gene (stx1) was amplified using PCR to evaluate the sample preparation method. Without prior enrichment of cells in broth media, the detection limit was 103 CFU g−1 beef. When a 6 h enrichment step was incorporated, the detection limit was 1 CFU g−1 beef. The total time required from beginning to end of the procedure was 12 h.
Conclusions: The sample preparation method developed here enabled substantially improved sensitivity in the PCR-based detection of E. coli O157:H7 in ground beef, as compared to previous reports.
Significance and Impact of the Study: Superb sensitivity, coupled with quick turn-around time, relative ease of use and cost-effectiveness, makes this a useful method for detecting E. coli O157:H7 in ground beef.