Rt pcr master mix

In vitro cultivation studies were performed using the following Actinomyces isolates: A. naeslundii (DSM-43013), A. oris (DSM-23056), A. johnsonii (DSM-23038), A. massiliensis (DSM-23047), A. gerencseriae (DSM-6844), A. dentalis (DSM-19115), and A. israelii (DSM-43320). Isolates were grown anaerobically in Schaedler Anaerobe Broth (SAB) medium in the presence of 0, 0.25, 0.5, 1, and 2% Maltitol. Cultivations were done in triplicate. Qualitative growth was assessed daily by visual inspection. Quantitative growth was assessed after 7 days of incubation by qPCR using primers 16S-Actmycs-gr-F 5′-GGGTTGTGAACCTCTTTCGCC-3′, 16S-Actmycs-gr-R 5′-GCTGGCACGTAGTTAGCCG-3′, and Taqman MGB probe 16S-Actmycs-gr 5′-TGTGGKKGGGTTGACG-3′. qPCR was performed in RT PCR master mix (Diagenode, Seraing, B) on an Applied Biosystems 7500 RT PCR system during 45 cycles of with a denaturation step at 95.0°C for 15 s and an annealing/elongation step at 60.0°C for 1 min. A standard curve was established by analysis of an A. massiliensis genomic DNA sample, serially diluted at 2 ng, 200 pg, 20 pg, 2 pg, 200 fg, 20 fg, and 2 fg per μl as well as a negative control.

For DNA isolation, fecal samples were thawed on ice and lysed by bead beating (Mini-BeadBeater-24, Biospec Products Bartlesville, USA) for 2 min at 2800 oscillations minute^− 1 in the presence of 300 μl of lysis buffer (Mag Mini DNA Isolation Kit, LGC ltd, UK), 500 μL zirconium beads (0.1 mm; BioSpec products, Bartlesville, OK, USA) and 500 μL phenol saturated with 10 mM Tris-HCl and 1 mM EDTA pH 8.0 (Sigma). After centrifugation DNA was extracted using the Mag Mini DNA Isolation Kit (LGC ltd, UK) in accordance to the manufacturers recommendations. DNA quality was assessed by routine gel electrophoresis as well as by capillary electrophoresis on the Fragment Analyzer (Advanced Analytical, Heidelberg, Germany). Quantitative PCR (qPCR) primers and probes are listed in Additional file 3: Table S1. qPCR was performed using RT PCR master mix (Diagenode, Seraing, B) on an Applied Biosystems 7500 RT PCR system.

Quantitative assessment of C. albicans abundance was performed by quantitative PCR using primers listed in Supplementary Table S7^{53 (link)}. qPCR was performed in RT PCR master mix (Diagenode, Seraing, B) on an Applied Biosystems 7500 RT PCR system during 40 cycles of with a denaturation step at 95.0 °C for 15 s and an annealing/elongation step at 60.0 °C for 1 min. A standard curve was established by analysis of genomic DNA of C. albicans ATCC 90,028. Cell densities were estimated by accounting for the elution volume used after DNA extraction (60 μl) and assuming a (diploid) genome size of 14 Mb for C. albicans. C. albicans abundance was then categorized into 3 groups: no C. albicans present, low C. albicans abundance (0 counts through the median number of counts), and high C. albicans abundance (the median number of counts through the highest number of counts).