The use of activated charcoal for the removal of PCR inhibitors from oyster samples.
Activated charcoal is a carbonaceous adsorbent with a high internal porosity, and hence a large internal surface area. Cells of a strain of Escherichia coli O157:H7 seeded into oyster tissue homogenates were completely bound to untreated charcoal after an incubation period of 15 min at room temperature. In contrast, activated charcoal particles coated with cells of Pseudomonas fluorescens resulted in 92.6%+/-3.7 recovery of E. coli O157:H7. This allowed the successful use of the coated activated charcoal for the absorption of PCR inhibitors from seeded tissue samples. With coated charcoal, real-time PCR was able to detect 1x10(3) CFU of E. coli 0157:H7/g of tissue which was equivalent to 50 genomic targets per real-time PCR. In contrast, without the use of treated charcoal, the real-time PCR failed to detect 10(7) CFU/g. This is a promising, and convenient technology that can be applied to increase the sensitivity of the PCR assay without selective enrichment, for the detection of low numbers of pathogenic microorganisms in complex matrices such as foods, clinical, and environmental samples, which frequently exhibit high levels of PCR inhibition.
Abolmaaty
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Department of Food Science, Massachusetts Agricultural Experiment Station, University of Massachusetts, Amherst, MA 01003, USA.
Gu
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Witkowsky
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Levin
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D000818:Animals; D002606:Charcoal / Q000494:pharmacology*; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004927:Escherichia coli Infections / Q000517:prevention & control; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D005516:Food Microbiology; D010125:Ostreidae / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D011551:Pseudomonas fluorescens / Q000378:metabolism*
D004269:DNA, Bacterial; D002606:Charcoal