Open AccessMethods for optimizing DNA extraction before quantifying oral bacterial numbers by real‐time PCR
Author(s) -
Nadkarni Mangala A.,
Martin F. Elizabeth,
Hunter Neil,
Jacques Nicholas A.
Publication year - 2009
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2009.01629.x
Subject(s) - bacteria , fusobacterium nucleatum , lactobacillus acidophilus , microbiology and biotechnology , biology , dna extraction , prevotella , fusobacterium , anaerobic bacteria , porphyromonas gingivalis , fastidious organism , lactobacillus , bacteroides fragilis , primer (cosmetics) , dna , polymerase chain reaction , bacteroides , chemistry , biochemistry , genetics , gene , probiotic , organic chemistry
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 °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.