Isolation and processing of dental plaque samples.

Plaque samples were placed in a sterile tube containing 2 mL phosphate-buffered saline (PBS) buffer and 10% glycerol as a cryoprotective agent, and the sample was stored at −80°C until processing. Upon thawing, 0.5 mL of the sample was subjected to lysis and chromosomal DNA purification using a Qiagen blood and tissue DNA purification kit with the modification that after resuspending the plaque sample in buffer ATL, digestion with proteinase K and addition of buffer AL, 500 μL of sterile zirconia beads (Fisher Scientific) were added, and the samples were sonicated for 2.75 min prior to proceeding with column purification of the extracted DNA (Mini Bead Beater; BioSpec Products). Purified DNA was quantified and used as a template for PCR analysis with 16S rRNA gene primers designed to detect all bacteria (universal primers flanking the V6 region) or detect each organism of interest (species-specific primers within the V6 region) including Streptococcus mutans/sobrinus, Actinomyces naeslundii/viscosus, Lactobacillus casei, or more diverse Lactobacillus species, Selenomonas species, Olsenella uli, Streptococcus sanguinis, Veillonella parvula/dispar, Prevotella multisaccharivorax, Prevotella denticola, Propionibacterium acidifaciens, Capnocytophaga granulosa, and Delftia acidovorans. Primers used for quantitative PCR are found in Table S1 in the supplemental material. Quantification of the level of each organism in plaque samples was determined using real-time PCR (Bio-Rad; CFX96) and comparison to a positive-control DNA standard for each organism of interest (Table S1). Reaction mixtures contained 10 μL SYBR Green supermix (Bio-Rad; catalog no. 1708882), 0.05 μL of each species primer (from 50 μM stock), 1 μL template DNA (≤1 ng/μL), and 9 μL H₂O. Fifty cycles of amplification were performed with 10 s at 95°C (melting), 30 s at 52°C (annealing), and 30 s at 72°C (elongation). PCRs were performed in duplicate wells on at least 3 separate days.