Ssoadvanced universal sybr green supermix 2

The expression patterns of eight randomly selected genes encoding the identified metabolites were analysed with primer pairs (Table 1) designed with the Primer 5 program (Premier Biosoft International, California, USA). cDNA was synthesised using 2 μg of total RNA (DNA-free), RevertAid reverse transcriptase (Thermo Scientific, Waltham, MA, USA) and Random Hexamer Primers (5′-NNNNNN-3′; N = G, A, T or C) (Thermo Scientific, Waltham, MA, USA) following the manufacturer’s protocol. The qPCR assay was conducted on a Rotor-Gene Q System (Qiagen, Hilden Germany) based on a method described previously^{51 (link),52 (link)} using SsoAdvanced Universal SYBR Green Supermix (2×) (Bio-Rad, Hercules, California, USA). The mixtures were subjected to an initial step at 95 °C for 1 min, followed by 40 cycles of denaturation at 95 °C for 20 s, annealing at 60.5 °C for 20 s, and elongation at 72 °C for 15 s. Melting curve analysis was performed by heating the amplicon from 72 °C to 95 °C. Relative gene expression levels were calculated according to the 2-ΔΔCT method, with ef1-α and rpl2 for T. rubrum, and adp-rf and mbp-1 for M. canis (Table 1) as the reference genes according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines^{56 (link)}.

To test the specificity of in silico selected primers, a generic quantitative PCR was carried out with a panel of reference DNAs. Primer and probes were obtained from Metabion (Planegg, Germany) (Table 5). The SYBR^® Green qPCR was performed in a total volume of 20 µL. For the PCR reaction mixture, 10 µL SsoAdvanced universal SYBR^® Green supermix (2×) (Biorad Laboratories GmbH, Munich, Germany), 10 pmol of each primer (forward and reverse), ultrapure nuclease-free water (Sigma-Aldrich, St. Louis, MO, USA) and 5 µL of template DNA were used. The cycling conditions in the SYBR^® Green real-time PCR were 98.0 °C (3 min, activating of Taq polymerase), followed by 40 cycles at 95.0 °C for 15 s and at 60.0 °C for 30 s. After each cycle, the light emitted by the fluorophore was measured. The melting curve was constructed from 65 to 95 °C at 0.5-°C increments with a dwell time of 5 s at each temperature. Real-time PCR results were analysed using the CFX Maestro software suite (Version: 3.1.15; Biorad Laboratories GmbH, Munich, Germany).

Total bacteria and potentially pathogenic species were quantified using qRT-PCR (CFX Connect Optics Module; Bio-Rad, Hercules, CA, USA) to determine bacterial loads. The PCR primers used are listed in Supplementary Table S1. PCR reaction mixtures were assembled from 10 μL SSoAdvanced universal SYBR Green supermix (2×; Bio-Rad), 10 μM each primer, and 1 μL metagenome DNA template or distilled water (negative control) in a final volume of 20 μL. PCR cycling was performed at 98 °C for 3 min (preheating), 98 °C for 15 s (denaturation), 56 °C for 30 s (annealing), 72 °C for 30 s (extension), and 72 °C for 5 min (final extension) in the CFX connect real-time PCR Detection system (Bio-Rad). Genomic DNA (gDNA) of pathogenic strains was extracted using the NucleoSpin Microbial DNA kit (Macherey-Nagel, Düren, NRW, Germany). The standard curves were generated using 10-fold dilutions of this bacterial gDNA (10² to 10⁶). The cycle threshold (Ct) values were determined after adjustment of the baseline (1000) using the CFX Manager software (BioRad, Hercules, CA, USA). The bacterial loads were quantified by comparing their Ct value to the standard curve. The regression coefficient (r²) value of the standard curve was greater than or equal to 0.99. All DNA samples were analyzed in triplicate.