Introduction Materials and methods Study sites and sampling
Fig. 1. Map indicating the geographic location of 21 Swiss Lakes sampled for our survey (Abbreviations of lake names are explained in Table 1. The map was created with ArcGIS (ESRI®ArcMap 10, Redlands, CA). 1.5-Column fitting image.Figure optionsDownload full-size imageDownload as PowerPoint slide
2.2. DNA extraction
2.3. qPCR assays
DNA extracts were screened for abundance of five different 10-DEBC genes (sul1, sul2, tet(B), tet(M), tet(W) and qnrA) using quantitative real-time PCR. Additionally, bacterial 16S rRNA gene fragments were quantified for normalizing resistance gene copy numbers per sample to the bacterial population size of each lake sample. Standard dilutions of plasmids containing the respective target genes were prepared in a range of 30 to 3 × 106 copies per 5 μl as described previously ( Czekalski et al., 2012).
Samples were considered as not quantifiable for a target gene if the standard deviation (SD) of Ct values of the filter triplicates was greater than 0.5 (unquantifiable sample) OR if the mean Ct value of the triplicates was equal to or greater than the mean Ct value of the first standard dilution with a SD of Ct values greater than 0.5 (below limit of quantitation). Samples were considered as below detection if their mean Ct value was greater than that of the lowest Ct value obtained from any negative controls for lipases assay OR if for 2 or more out of 3 Ct values of the replicates were not recorded (Czekalski et al., 2014). Limits of quantitation and detection for each qPCR assay are summarized in Table A.1 of Appendix A).