Journal
FEMS MICROBIOLOGY LETTERS
Volume 296, Issue 1, Pages 45-51Publisher
OXFORD UNIV PRESS
DOI: 10.1111/j.1574-6968.2009.01629.x
Keywords
DNA extraction; nuclease activity; oral bacteria; real-time PCR
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Funding
- NIDCR [R01 DE015272-07]
- Dental Board of New South Wales, Australia
- Institute of Dental Research
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Methods for the optimal extraction of genomic DNA for real-time PCR enumeration of oral bacteria using the muramidase, mutanolysin, were developed using a simple in vitro oral flora model comprised of the facultative anaerobic Gram-positive bacteria, Lactobacillus acidophilus and Streptococcus mutans, the Gram-positive anaerobe, Parvimonas micra, and the Gram-negative anaerobes, Porphyromonas gingivalis, Prevotella melaninogenica and Fusobacterium nucleatum. Traditional, as well as more elaborate, methods of quantifying bacterial numbers, including colony counting and estimation of DNA content using 4',6-diamino-2-phenylindole were compared in order to validate the real-time PCR approach. Evidence was obtained that P. gingivalis nuclease activity adversely affected the extraction of double-stranded DNA from this bacterium either alone or when it formed part of a consortium with the other bacteria. This nuclease activity could be overcome by treatment of the bacteria with either 20 mM diethyl pyrocarbonate or 70% ethanol at 4 degrees C overnight. A final purification of the DNA to remove any potential PCR inhibitors was added to the protocol in order to accurately quantify the amount of DNA by real-time PCR and hence the number of bacteria in a sample.
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