MagnaExtract, a novel magnetic bead-based extraction method for the molecular detection of antimicrobial resistance genes in fresh water.
BackgroundThe environmental is increasingly recognised as an important reservoir of antimicrobial resistance (AMR) genes. Polymerase chain reaction (PCR) and whole genome sequencing (WGS) have great potential in the surveillance of AMR genes. However, molecular methods are dependent upon the isolation of high-quality DNA yields. Currently, there is no consensus for the optimum DNA extraction strategies from complex environmental matrices for downstream molecular applications. MethodsWe present a novel magnetic bead-based method for the isolation of antimicrobial resistance genes (ARGs) from river water in Malawi, named MagnaExtract. We present this with analytic limit of detection (LOD) as well as a case study in Southern Malawi. Here we compare the DNA yield and subsequent PCR output from MagnaExtract with commercially available QIAGEN kits and the crude boil and spin method, utilising a high-resolution melt analysis (HRM) PCR panel designed for the detection of third generation cephalosporin and carbapenem resistant genes. ResultsOf the 98 water samples evaluated we found the MagnaExtract method to be comparable, and in some instances superior to commercially available kits for the isolation of ARGs from river water samples. In addition, we found overnight incubation to promote the recovery of extended spectrum beta-lactamase (ESBL) genes and simultaneous reduction in the detection of carbapenemase genes. ConclusionThe MagnaExtract approach offers a simple, affordable, high yielding extraction method that could be used for the detection of ARGs isolated from river water samples in environmental surveillance campaigns in East Africa.