*3.5. Quantitative PCR*

Quantitative PCR was used to analyze selected genes coding for resistance to tetracyclines and sulfonamides in the collected samples. The 16S rRNA gene was used as the reference gene to account for variability of total amount of bacteria in the samples. Primer sequences adapted from other studies [31,42,77,78] or designed for this study (*tetM*) are listed in Table 5. All qPCR assays were performed in the Roche LightCycler ® 480 II (Roche Applied Science, Indianapolis, IN, USA) in 11 μL reaction mixture. Analyzes for each sample were carried out in triplicate. PCR mixtures consisted of 5-μL LightCycler 480 SYBR Green I Master (Roche Applied Sciences, Indianapolis, IN, USA), 2.5 μL each primer of corresponding concentration 5 μM (*tetA, B, C, L, M, O, X, sulI*, *II*), 2 μM (*tetG, K, sulIII*), 1 μM (*tetQ*) and 1 μL DNA template of 5 ng/μL. In each run, 1 μL microbial DNA-free water as negative control was included. The thermal cycling conditions selected for studied genes are given in Table 6. Specificity of the amplified PCR product was verified by performing melting curve analysis, while amplification e fficiency was assessed by monitoring the slope of amplification curves generated for the target genes and the internal controls (16S rRNA). Quantitative analysis of qPCR data was carried out using a comparative CT method (also known as ΔΔ CT, or <sup>2</sup>−ΔΔCT) based on the literature [79]. In the first stage, threshold cycles (CT) of the tested ARGs and control gene (16S rRNA) amplification reactions in all samples were determined. The CT value is provided as the result of qPCR analysis for each gene. In turn, for individual samples (raw influent and final e ffluent), the di fferences between the CT values for the tested gene and 16S rRNA were calculated according to Formula (1). The obtained results present the ARG abundance relative to that of 16S rRNA. Then the ΔΔ CT values for each gene were obtained with the use of Formula (2). Computation the normalized value of the relative level of the tested gene in the unknown sample (final e ffluent) relative to the calibrator sample (raw influent) was carried out based on the Formula (3). The value of parameter R equal to 1 means the relative amount of gene in both samples is comparable, <1 indicates a decrease in the relative amount of the gene in the final wastewater sample, while >1 shows the enrichment of the gene after the wastewater treatment process.

$$
\Delta C\_{\text{T (influence(effluxrt)}} = C\_{\text{T (ARG)}} - C\_{\text{T (Id\& rRNA)}} \tag{1}
$$

$$
\Delta\Delta\text{CT} = \Delta\text{CT}\_{\text{(effluent)}} - \Delta\text{CT}\_{\text{(influence)}} \tag{2}
$$

$$\mathcal{R} = \mathcal{Z}^{-\text{\textquotedblleft ACT}} \tag{3}$$


**Table 6.** qPCR conditions used in this study.
