title: Methods for optimizing DNA extraction before quantifying oral bacterial numbers by real-time PCR.
abstract: 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.
authors.affiliation: Institute of Dental Research, Westmead Millennium Institute and Westmead Centre for Oral Health, Wentworthville, NSW, Australia. mangala_nadkarni@wmi.usyd.edu.au
authors.forename: Mangala A
authors.initials: MA
authors.lastname: Nadkarni
authors.forename: F Elizabeth
authors.initials: FE
authors.lastname: Martin
authors.forename: Neil
authors.initials: N
authors.lastname: Hunter
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Jacques
mesh_terms: D002473:Cell Wall / Q000378:metabolism; D015169:Colony Count, Microbial / Q000379:methods*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology; D009055:Mouth / Q000382:microbiology*; D009113:Muramidase / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D000431:Ethanol; D003851:Deoxyribonucleases; D009113:Muramidase; D010450:Endopeptidases; C021816:mutanolysin; D004047:Diethyl Pyrocarbonate