title: Detection of 5 CFU/g of Escherichia coli O157:H7 on lettuce using activated charcoal and real-time PCR without enrichment.
abstract: A sample treatment method which separates Escherichia coli O157:H7 from lettuce and removes PCR inhibitors allowing 5 CFU/g of target cells to be detected using real-time PCR is described. Lettuce leaves inoculated with E. coli O157:H7 were rinsed with 0.025% sodium dodecyl sulfate (SDS). In this study, there were two major factors that strongly affected the recovery of E. coli O157:H7 during sample preparation, the amount of bentonite coated activated charcoal used to remove PCR inhibitors and the agitated contact time of the samples with the coated charcoal. When 3.0 g of activated carbon coated with bentonite were mixed with target cell suspensions (30 ml) derived from 50 g of lettuce, a high recovery of E. coli O157:H7 (93%) was obtained. Sample agitation with bentonite coated activated charcoal for 15 min resulted in 95% recovery of E. coli O157:H7. When a commercial DNA purification resin was used for detection of E. coli O157:H7 without the use of the bentonite treated charcoal, the real-time PCR (Rti-PCR) failed to detect 1 × 10(2) CFU/g. In contrast, with the use of use of bentonite coated activated charcoal and a commercial DNA purifying resin together, Rti-PCR was able to detect 5 CFU of E. coli O157:H7/g of lettuce which was equivalent to 2.8 CFU/Rti-PCR. Such a successful detection level was the result of the bentonite coated activated charcoal's ability to absorb the PCR inhibitors released from seeded lettuce during detachment. A standard curve was generated by plotting the Ct values against the log of CFU of target bacterial cells. A linear range of DNA amplification was exhibited from 5.0 × 10(0) to 1.0 × 10(4) CFU/g by using Rti-PCR.
authors.affiliation: Department of Human Ecology, College of Agriculture & Related Sciences, Delaware State University, Dover, DE 19901, USA.
authors.forename: Jung-Lim
authors.initials: JL
authors.lastname: Lee
authors.forename: Robert E
authors.initials: RE
authors.lastname: Levin
mesh_terms: D001546:Bentonite; D002606:Charcoal; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis*; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D005506:Food Contamination / Q000032:analysis*; D005784:Gene Amplification; D018545:Lactuca / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D001546:Bentonite; D002606:Charcoal