25 U of Taq DNA polymerase (Fermentas), and 0.2 mmol/L of each deoxyribonucleoside
triphosphate (Biotools, Madrid, Spain). Positive and negative controls were included in each batch of samples analyzed. Positive controls consisted CP-868596 nmr of DNA extracted from Porphyromonas gingivalis (ATCC 33277), Methanobrevibacter arboriphilus (DSMZ 744), and Candida albicans (ATCC 10231). Negative controls consisted of sterile ultrapure water instead of sample. All reactions were run in triplicate. PCR amplifications were performed in a DNA thermocycler (Mastercycler personal; Eppendorff, Hamburg, Germany). Cycling conditions were as follows. For bacteria, it included initial denaturation step at 95°C for 2 minutes, followed by 36 cycles at 95°C/30 seconds, 60°C/1 minute, and 72°C/1 minute, and final extension at 72°C/10 minutes. For archaea, it included initial denaturation at 94°C/2 minutes, 36 cycles at 94°C/30 seconds, 58°C/30 seconds, and 72°C/1 minute, and final extension at 72°C/10 minutes. For fungi, it included initial denaturation step at 95°C/30 seconds, followed by 40 cycles at 95°C/30 seconds, 55°C/1 minute, 72°C/2 minutes, and a final step at 72°C/10 minutes. PCR products were subjected to electrophoresis in a 1.5% agarose gel–Tris-borate-EDTA
buffer. The gel was stained with GelRed (Biotium, Hayward, CA) and visualized under ultraviolet Akt inhibitor illumination. The presence of amplicons of the expected size for each primer pair was considered as positive result. A 100 base pair DNA ladder (Biotools) was used as a parameter for amplicon size. For bacterial identification in the checkerboard assay, a practically
full-length 16S rRNA gene fragment was amplified by using universal bacterial primers 8f and 1492r, with the forward primer labeled at the 5′ end with digoxigenin. PCR amplifications were performed as Metformin cost described above for bacteria. The reverse-capture checkerboard assay was conducted to determine the presence and levels of 28 bacterial taxa as described previously 20, 24 and 25. Probes were based on 16S rRNA gene sequences of the target bacteria and were described and validated elsewhere 20, 24, 26 and 27. Prevalence of the target taxa was recorded as the percentage of cases examined. A semiquantitative analysis of the checkerboard findings was conducted as follows. The obtained chemiluminescent signals were evaluated by using ImageJ (W. Rasband, http://rsb.info.nih.gov/ij/) and converted into counts by comparison with standards at known concentrations run on each membrane.