Development of a rapid field-applicable molecular diagnostic for knockdown resistance (kdr) markers in An. gambiae

Abstract

BackgroundThe spread of insecticide resistance (IR) is a major threat to vector control programmes for mosquito-borne diseases. Early detection of IR using diagnostic markers could help inform these programmes, especially in remote locations where gathering reliable bioassay data is challenging. Most current molecular tests for genetic IR markers are only suitable for use in well-equipped laboratory settings. There is an unmet need for field-applicable diagnostics.MethodsA single-cartridge test was designed to detect key IR mutations in the major African vector of malaria, Anopheles gambiae. Developed on the portable, rapid, point-of-care compatible PCR platform - Genedrive® (genedrive® plc), the test comprises two assays which target single nucleotide polymorphisms (SNPs) in the voltage gated sodium channel (VGSC) gene that exert interactive effects on knockdown resistance (kdr): L1014F, L1014S and N1575Y.ResultsDistinct melt peaks were observed for each allele at each locus. Preliminary validation of these assays using a test panel of 70 An. gambiae samples showed complete agreement of our assays with the widely-used TaqMan assays, achieving a sensitivity and specificity of 100%.ConclusionHere we show the development of an insecticide resistance detection assay for use on the Genedrive® platform that has the potential to be the first field-applicable diagnostic for kdr.

Development and application of a tri-allelic PCR assay for screening Vgsc-L1014F kdr mutations associated with pyrethroid and organochlorine resistance in the mosquito Culex quinquefasciatus

“…Several diagnostic tests for typing kdr mutations have already been developed and applied in Culex , Anopheles and Aedes species [20–22]. Whilst many rely on standard PCR methods [23–26], over the past few years there has been an increase in the use of high-throughput methods such as quantitative PCR (e.g.…”

“…Regardless of methodology, most assays are designed to perform bi-allelic discrimination, and there are a limited number of low-cost approaches for typing tri-allelic mutations (e.g. [20, 24, 28]) such as the alternative codons (TTA/TTT/TTC) at locus Vgsc -1014 already reported in C. quinquefasciatus populations from Africa and Asia [18, 21, 29].…”

Development and Application of a Tri-allelic PCR Assay for ScreeningVgsc-L1014FKdrMutations Associated with Pyrethroid and Organochlorine Resistance in the MosquitoCulex quinquefasciatus

“…Several diagnostic tests for typing kdr mutations have already been developed and applied in Culex, Anopheles and Aedes species (20)(21)(22). Whilst many rely on standard PCR methods (23)(24)(25)(26), over the past few years there has been an increase in the use of high-throughput methods such as quantitative PCR (e.g.…”

“…Regardless of methodology, most assays are designed to perform bi-allelic discrimination, and there are a limited number of low cost approaches for typing tri-allelic mutations (e.g. (20,24,28) such as the alternative codons (TTA/TTT/TTC) at locus Vgsc-1014 already reported in C. quinquefasciatus populations from Africa and Asia (18,21,29).…”