QuickLyse Miniprep Kit (250)

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Publication protocol

Experiment 1—Comparison of DNA extraction methods: Preliminary experiments were performed to identify a DNA extraction protocol that would efficiently extract resistance genes present in Enterobacteriaceae. As acquired resistance genes in Enterobacteriaceae are typically present on plasmids, we hypothesised that a plasmid extraction protocol would increase detection of these genes. The QuickLyse Miniprep Kit (Qiagen), a spin column-based plasmid extraction kit, was compared to a standard fecal DNA extraction protocol, the QIAamp Fast DNA Stool Mini Kit (Qiagen) modified to include mechanical lysis with bead beating. One hundred milligram aliquots from two different volunteer fecal sample were spiked with one of two Enterobacteriaceae carrying plasmid-associated beta-lactamases (K. pneumoniae with NDM-1 and CTX-M-15, or E. cloacae with KPC-2 and TEM-1). Spike organisms were added at a concentration of 108 CFU/g and negative controls with no spike were run in parallel. Following DNA extraction using the two methods, sequencing was performed on Illumina MiSeq with a mean sequencing depth of 1.4 million paired reads per sample.



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