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

• Published in: Parasites & vectors Vol. 12; no. 1; pp. 232 - 6
• Main Authors: Silva Martins, Walter Fabricio, Silva Pereira, Bárbara Natieli, Vieira Alves, Ana Thayse, Murphy, Annabel, Silva Martins, Paulo Geovani, Weetman, David, Wilding, Craig Stephen, Donnelly, Martin James
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 14.05.2019; BioMed Central; BMC
• Subjects: Alleles; Allelic-specific PCR; Analysis; Animals; Aquatic insects; Assaying; Binding sites; Biological Assay; Chlorine compounds; Codons; Culex - genetics; Culex quinquefasciatus; Culicidae; DDT; Design; Detection; Diagnostic systems; Disease transmission; Diseases; Distribution; DNA; Female; Filariasis; Genotyping; Genotyping Techniques; Hydrocarbons, Chlorinated; Insect carriers of disease; Insecticide resistance; Insecticide Resistance - genetics; Insecticides; Leucine; Mosquitoes; Mutation; Nucleotide sequence; Organic chlorine compounds; Organic compounds; Organochlorine pesticides; PCR; Phenylalanine; Polymerase Chain Reaction; Public health; Pyrethrins; Pyrethroids; Regions; Screening; Sodium; Sodium channels (voltage-gated); Specificity; Sustainability; Sustainable development; Target-site mutations; Tropical climate; Tropical environment; Tropical environments; Uganda; Varieties; Vector-borne diseases; Voltage-Gated Sodium Channels - genetics; West Nile virus; Uganda; Brazil; Tanzania; United States > US; China; Vector-borne diseases; Allelic-specific PCR; Target-site mutations
• ISSN: 1756-3305; 1756-3305
• DOI: 10.1186/s13071-019-3490-z

Culex quinquefasciatus has a widespread distribution across tropical and sub-tropical regions, and plays an important role in the transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF) and multiple arboviruses. Increased resistance to insecticides threatens the efficacy and sustainability of insecticide-based anti-vector interventions which mitigate the burden of mosquito transmitted diseases in endemic regions. In C. quinquefasciatus two non-synonymous voltage gated sodium channel (Vgsc) variants, both resulting in a leucine to phenylalanine change at codon 1014, are associated with resistance to pyrethroids and DDT. This tri-allelic variation has compromised the ability to perform high-throughput single-assay screening. To facilitate the detection and monitoring of the Vgsc-1014 locus in field-caught mosquitoes, an Engineered-Tail Allele-Specific-PCR (ETAS-PCR) diagnostic assay was developed and applied to wild mosquitoes from Brazil, Tanzania and Uganda. This new cost-effective, single-tube assay was compared to two, well-established, genotyping approaches, pyrosequencing and TaqMan. The ETAS-PCR assay showed high specificity for discriminating the three alleles at Vgsc-L1014F, with genotyping results strongly correlated with pyrosequencing and TaqMan results (98.64% and 100% respectively). Our results support the utility of the ETAS-PCR/Vgsc-1014 diagnostic assay, which stands as an effective alternative for genotyping tri-allelic variants.