Use of activated carbon coated with bentonite for increasing the sensitivity of pcr detection of Escherichia coli O157:H7 in Canadian oyster (Crassostrea gigas) tissue.
A novel method for directly increasing the recovery of Escherichia coli O157:H7 and efficiently eliminating PCR inhibitors in oyster tissue without preenrichment was developed with the use of activated carbon coated with bentonite. The recovery of E. coli O157:H7 was significantly affected by the amount of bentonite used to coat the activated charcoal and the pH value of sample preparations. When 4.2 g of activated carbon were coated with 0.4 g of bentonite and seeded oyster samples were adjusted to a pH of 5.0, a high recovery of E. coli O157:H7 (91.6+/-4.4%) was obtained. Activated carbon, coated with bentonite, allowed the PCR detection of 1.5 x 10(2) CFU/g of oyster tissue which was equivalent to 30 genomic targets per PCR reaction. Without the use of activated carbon coated with bentonite, the minimum level of detection was 1.5 x 10(5) CFU/g of oyster tissue, which is equivalent to 3.0 x 10(4) genomic targets per PCR reaction. Three commercial DNA purification systems were used for comparison. The limit of detection with the Wizard DNA Clean-Up System and the Chelex(R)100 Resin was 1.5 x 10(3) CFU/g of oyster tissue which was equivalent to 3.0 x 10(2) CFU/PCR reaction. The QIAamp DNA Mini Kit resulted in a detection limit of 5 x 10(2) CFU/g of oyster tissue which was equivalent to 5 x 10(2) genomic targets per PCR reaction. The use of activated carbon coated with bentonite is an inexpensive method for removal of PCR inhibitors from tissue samples prior to the release of DNA from target cells resulting in relatively low numbers of target cells detected without enrichment.
Department of Food Science, Massachusetts Agricultural Experiment Station, University of Massachusetts, Amherst, MA 01003, USA.
Chunguang
C
Luan
Robert E
RE
Levin
D000818:Animals; D001431:Bacteriological Techniques; D001546:Bentonite; D002170:Canada; D002606:Charcoal; D049896:Crassostrea / Q000382:microbiology*; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic; D001546:Bentonite; D002606:Charcoal