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bioRxiv · 10.1101/588905

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

Silva Martins, W. F.·Silva Pereira, B. N.·Vieira Alves, A. T.·Murphy, A.·Silva Martins, P. G.·Weetman, D.·Wilding, C. S.·Donnelly, M. J.
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Abstract

BackgroundCulex 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.\n\nResultsThis 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 98.64% and 100% against pyrosequencing and TaqMan, respectively.\n\nConclusionsOur results support the utility of the ETAS-PCR/Vgsc-1014 diagnostic assay, which stands as an effective alternative for genotyping tri-allelic variants.

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Silva Martins, W. F., Silva Pereira, B. N., Vieira Alves, A. T., Murphy, A., Silva Martins, P. G., Weetman, D., Wilding, C. S., Donnelly, M. J.. 2019-03-26. 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. https://doi.org/10.1101/588905

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