Detection of a single nucleotide polymorphism for insecticide resistance using recombinase polymerase amplification and lateral flow dipstick detection

Modern methods of single nucleotide polymorphism (SNP) detection require high-throughput or automated technologies that preclude field deployment. To develop a low-resource SNP detection system, we evaluated Recombinase Polymerase Amplification coupled with lateral flow dipstick (RPA-LFD) detection, targeting, as a proof-of-concept, an SNP (F1534C) that leads to pyrethroid resistance in Aedes aegypti mosquitoes. Primers and probes were designed to contain variable numbers and positions of mismatches and tested for their ability to amplify synthetic gene fragments with either wild-type or the single mismatch mutant gene sequence. Our results demonstrated, for the first time, that detection of a single nucleotide change could be achieved in this low-resource format by performing tenfold serial dilutions of gene fragments, which reduced non-specific amplification at high template concentrations. Using a forward primer SNP detection approach, removing the reverse primer enhanced discrimination with single, double and three scattered mismatches, with sensitivity decreasing 10-fold per additional mutation; a single 3′ mismatch provided the greatest (3-log) difference in sensitivity between the two variants. Similar results were observed using a reverse probe-based mismatch approach, however, the forward primer variants showed greater impacts on RPA efficiency. These results demonstrate significant potential for SNP mutant detection using a rapid 15-minute low-resource test.