bA real-time PCR assay developed to quantify Methanobrevibacter oralis indicated that its inoculum significantly correlated with periodontitis severity (P ‫؍‬ 0.003), despite a nonsignificant difference in prevalence between controls (3/10) and patients (12/22) (P ‫؍‬ 0.2, Fisher test). The M. oralis load can be used as a biomarker for periodontitis. Periodontitis is an anaerobic infection possibly leading to loss of teeth (1-3). It likely results from infection with microbial complexes comprising methanogens (4, 5), mostly Methanobrevibacter oralis (6)(7)(8). In this study, we correlated the M. oralis load as measured by real-time PCR in subgingival plaque with the severity of periodontitis.All patients diagnosed with periodontitis from October 2011 to June 2012 were included. The control individuals with generally healthy gingiva were volunteers. All patients underwent interviews for medical and dental history, intraoral examination to determine bleeding on probing (BOP), probing depth (PD), and plaque index (PI), and radiographs (9); periodontal status was also scored as described previously (10) ( Table 1). All individuals signed an informed-consent form, and the ethics committee of the IFR 48, University of Aix-Marseille, approved the protocol.Subgingival plaque specimens (50 l) collected from 3-to 12-mm periodontal pockets in both patients and controls were suspended in 1 ml Tris-HCl (0.05 M, pH 7.5) buffer. After homogenization, a 250-l aliquot was shaken with 0.3 g of acid-washed beads (Յ106 mm; Sigma, Saint-Quentin-Fallavier, France) in a FastPrep-24 instrument (MP Biomedical Europe, Illkirch, France) at a speed of 6.5 m/s (full speed) for 90 s. The supernatant was incubated overnight at 56°C with 180 ml of lysis buffer and 25 l proteinase K (20 mg/ml) in the Qiagen EZ1 DNA tissue kit (Qiagen, Courtaboeuf, France). After a second cycle of mechanical lysis, the supernatant was incubated for 10 min at 100°C, and total DNA was then extracted using the same kit. Negative controls consisting of sterile DNA-free water were introduced in all the manipulations. The specificity of the M. oralis-cnp602P probe (6-carboxyfluorescein [FAM]-5=AGCAGTGCACCTGCTGATA TGGAAGG-3=) (Applied Biosystems, Courtaboeuf, France) and the primer pair M. oralis-cnp602F (5=-GCTGGTGTAATCGAAC CTAAACG-3=) and M. oralis-cnp602R (5=-CACCCATACCCGG ATCCATA-3=) (Eurogentec, Seraing, Belgium) was verified in silico using the BLAST program at NCBI (http://www.ncbi.nlm .nih.gov/BLAST) and further experimentally ensured by incorporating DNA extracted from Methanobrevibacter smithii ATCC 35061 T , Methanosphaera stadtmanae ATCC 43021 T , Methanomassiliicoccus luminyensis CSURP135 T , Escherichia coli, Salmonella enterica, Staphylococcus aureus, and Treponema denticola ATCC 35405T . Real-time PCR assays were performed with a CFX96 Touch real-time PCR detection system (Bio-Rad, Marnes-laCoquette, France) using the Mastermix PCR kit (Eurogentec) with 5 pmol of each primer and probe and 5 l of about 2 g of DNA into a 20-l final volume. M. oralis DSMZ ...