Methods for optimizing DNA extraction before quantifying oral bacterial numbers by real-time PCR

作者：Mangala A. Nadkarni, F. Elizabeth Martin, Neil Hunter, Nicholas A. Jacques · 发表于：FEMS Microbiology Letters · 年份：2009 · DOI：10.1111/j.1574-6968.2009.01629.x · 被引用次数：38 · 研究领域：Oral microbiology and periodontitis research、HIV/AIDS oral health manifestations、Probiotics and Fermented Foods

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.