Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef.
OBJECTIVE
To develop an improved, rapid and sensitive sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef.


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.


CONCLUSIONS
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.
Cui
S
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Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, USA. jm332@umail.umd.edu
Schroeder
C M
CM
Zhang
D Y
DY
Meng
J
J
D000818:Animals; D002417:Cattle; D002498:Centrifugation; D003470:Culture Media; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D005516:Food Microbiology; D005798:Genes, Bacterial / Q000235:genetics; D008460:Meat / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1 / Q000235:genetics
D003470:Culture Media; D022622:Shiga Toxin 1