Abstract：
: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.
journal_name
J Microbiol Methodsjournal_title
Journal of microbiological methodsauthors
Luan C,Levin REdoi
10.1016/j.mimet.2007.10.010subject
Has Abstractpub_date
2008-01-01 00:00:00pages
67-72issue
1eissn
0167-7012issn
1872-8359pii
S0167-7012(07)00353-3journal_volume
72pub_type
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