E. faecalis-specific

primers targeting azoA (encoding azoreductase; sense: Ef azoAF 5′-CCAATCAAATGGCGGCTTCTACG-3′, antisense: Ef azoAR 5′-GCGATCAGGGAAATGATCGATTCC-3′) were designed (11). Primer specificity was confirmed by PCR using chromosomal DNA from 28 oral bacteria (Table 1). SYBR green-based quantitative real-time PCR was performed in a total volume of 20 μL containing 5 μL of various concentrations of extracted genomic DNA with or without PMA treatment, 5 × SYBR Green Master (Roche Diagnostics, Mannheim, Germany), and 0.5 μM of each primer. Amplification was done using the LightCycler Carousel-Based System (Roche Diagnostics) at 95°C for 10 min, followed by 45 cycles of 95°C for 10 s, 53°C for 10 s, and 72°C for 12 s. To confirm the formation of a single product, melting curve analysis was performed at 95°C for 1 min Ibrutinib purchase and 55°C for 1 min, with a subsequent temperature increase from 55.0–95.0°C at 0.5°C per 10 s (data Vemurafenib not shown). The sizes of the products were confirmed using

2% agarose gels. Using this method, bacterial CFU were detected linearly from 15 to 3.0 × 107 per mixture. The relationship between live cells and Ct values for real-time PCR is as follows: Y = 10−0.293X±11.056 (where Y = log10CFU, X = Ct value, R2= 0.997). Bacterial cell numbers were calculated using this formula. Propidium monoazide (Biotium, Hayward, CA, USA) was dissolved in 20% DMSO to produce a 24 mM stock solution. Following incubation with the dye for 5 min in the dark, similarly prepared cells were exposed for 5 min to a 600 W halogen light placed 20 cm above 500 μL samples in open microcentrifuge tubes on ice. The toxicity of PMA at 2.4 μM to 2.4 mM to E. faecalis was analyzed at 37°C; however, no toxicity was found (Mann-Whitney U-test, data not shown). In this study, 240 μM of PMA was employed for the analysis. To investigate the effects of PMA, E. faecalis chromosomal DNA (0.01–100 μg/mL) was analyzed with and without PMA treatment. Real-time PCR was not inhibited by heat-killed cells treated FER with 240 μM PMA (Fig. 1). To eliminate possible inhibition by

the clinical material, E. faecalis samples were spiked with dental plaque and saliva (without E. faecalis) to mimic the oral environment. There was no inhibition of real-time PCR (Fig. 2). Based on these results, nine endodontic samples from eight patients with root-filled teeth and showing radiographic evidence of apical periodontitis were analyzed. The endodontic samples were collected in accordance with the guidelines of the Ethics Committee of Kyushu Dental College Hospital from patients who visited the Department of Preventive Dentistry, Kyushu Dental College Hospital. All patients provided informed consent. Endodontic samples were taken from the infected root canals as described previously (12). The relevant tooth was isolated from the oral cavity with a disinfected rubber dam.