Magnetic concentration of allele-specific products from recombinase polymerase amplification

Highlights: • Novel in-chip homogeneous hybridization of RPA products. • Magnetic concentration of products hybridized to probe-conjugated beads. • Point-of-care detection of mutant variants. • Accurate identification of single-nucleotide substitutions in tumour tissues. -- Abstract: The studied challenge is the specific detection of low-abundant genomic variants that differ by a single nucleotide from the wild type. The combination of blocked recombinase polymerase amplification (RPA) and selective capture by probes immobilised on magnetic-core particles integrated into a flow system is presented. The sensing principle was demonstrated as the effective concentration-detection of the specific generated products was achieved. The analytical performance of resulting assay was successfully compared to PCR-based methods or array formats, providing faster effective detection of the selective products. As proof of concept, the single-nucleotide substitutions of the KRAS gene at codon 12 were studied in chip with parallel microchambers and permanent magnets. The blocked RPA products (generated at 37 °C) from tumour biopsies (extracted DNA 4 ng) provided a specific fluorescent bead-line that depends on the present mutation. The results agree with those reported by next-generation sequencing and provide new opportunities for in vitro diagnostic and personalised medicine.