Efficiency and sensitivity of the digital droplet PCR for the quantification of antibiotic resistance genes in soils and organic residues

Abstract

Droplet digital PCR (ddPCR) allows absolute quantification and tolerance to inhibitors and has been proposed as the method of choice to overcome limitations of qPCR. The aim of this study was to evaluate ddPCR and qPCR performances to detect low copy number and copy number variation of antibiotic resistance genes (sul1 and qnrB genes encoding for resistance to sulfonamides and quinolones, respectively) using bacterial genomic DNA (gDNA) and metagenomic DNA extracted from soil and organic residue samples. With gDNA, qPCR showed a better range of quantification but the lower limit of quantification was at 15 copies of qnrB target vs. 1.6 in ddPCR. In the presence of background DNA or inhibitors, we observed a high loss of sensitivity in qPCR and an overestimation of target sequences. When using high amount of environmental DNA templates (70 ng per reaction), ddPCR was still allowing accurate quantification without adding PCR facilitator (i.e., T4 Gene 32 protein). Sensitivity to detect copy number variation was tenfold higher in ddPCR than in qPCR. Finally, the advantages of using ddPCR in environmental studies were confirmed with the quantification of sul1 and qnrB in soils, manures, or urban wastes.

Keywords: Antibiotic resistance gene; Digital droplet PCR; Organic residue; Soil.