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 × 102 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 × 105 CFU/g of oyster tissue, which is equivalent to 3.0 × 104 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®100 Resin was 1.5 × 103 CFU/g of oyster tissue which was equivalent to 3.0 × 102 CFU/PCR reaction. The QIAamp® DNA Mini Kit resulted in a detection limit of 5 × 102 CFU/g of oyster tissue which was equivalent to 5 × 102 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.