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.

Ecology of the microbiome of the infected root canal system: a comparison between apical and coronal root segments

“…Li et al. (2010) used Qiagen lysis buffer and proteinase K treatment (QIAamp DNA mini kit; Qiagen, Valencia, CA, USA), which has been shown to underestimate Gram‐positive bacteria compared with more easily lyzed Gram‐negative bacteria (Nadkarni et al. 2009).…”

“…The most likely reason, however, is because of differences in DNA extraction technique. Li et al (2010) used Qiagen lysis buffer and proteinase K treatment (QIAamp DNA mini kit; Qiagen, Valencia, CA, USA), which has been shown to underestimate Gram-positive bacteria compared with more easily lyzed Gram-negative bacteria (Nadkarni et al 2009). In the experience of the authors (unpublished findings), this particular DNA extraction method, if used according to the manufacturer's instructions, yields lower sample diversity than if an additional bead-beating step is added to enhance rupture of more resilient Gram-positive cells.…”

pH gradient and distribution of streptococci, lactobacilli, prevotellae, and fusobacteria in carious dentine

“…In a bacterial community, where dead and alive mixture of bacteria may present, the nuclease activity of bacteria will decompose the remnants of DNA, and hence, false overdetection and overquantification of dead bacteria is inherently minimized [23]. We carried out qPCR in triplicate in 25 µl volume reactions using Platinum ® quantitative PCR SuperMix-UDG (Invitrogen™) for TaqMan primer and probe sets and Platinum ® SYBR ® Green qPCR SuperMix-UDG (Invitrogen™) for the lactobacilli primer set, containing 200 µM of forward and reverse primers and 100 pg to 1 µg of genomic DNA template, as well as 100 µM of fluorogenic probe for TaqMan primer and probe sets.…”

Lactobacilli Are Prominent in the Initial Stages of Polymicrobial Infection of Dental Pulp

“…The sections were incubated with 0.2 M HCl for 10 min, washed with 18-M⍀ water, treated with 1% Triton X-100 for 90 s, and washed with 18-M⍀ water. The sections were then digested at 37°C with 2 mg proteinase K ml Ϫ1 , 2 mg lysozyme ml Ϫ1 , and 1,000 U mutanolysin ml Ϫ1 in 10 mM phosphate buffer, pH 6.7, for 40 min to facilitate penetration of the probe into the bacteria (17). The digestion was stopped with a 2-mg glycine ml Ϫ1 wash in 50 mM Tris buffer, pH 7.2.…”

“…DNA was extracted from 3 to 5 sections (2-m thickness each) of resin-embedded lesions 1 and 9 (see Table 3). Samples were incubated with cell lysis buffer as described previously (17). After addition of SDS (1%), DNA was extracted using the QIAamp DNA minikit (Qiagen) per the manufacturer's instructions, with a final elution volume of 200 l.…”