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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, WF, Silva Pereira, BN, Vieira Alves, AT, Murphy, A, Silva Martins, PG, Weetman, D, Wilding, CS and Donnelly, MJ (2019) 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. Parasites and Vectors, 12 (1). ISSN 1756-3305

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Abstract

Background: 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.
Results: 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).
Conclusions: Our results support the utility of the ETAS-PCR/Vgsc-1014 diagnostic assay, which stands as an effective alternative for genotyping tri-allelic variants.

Item Type: Article
Uncontrolled Keywords: 1108 Medical Microbiology, 1117 Public Health and Health Services
Subjects: Q Science > QR Microbiology
R Medicine > RA Public aspects of medicine > RA0421 Public health. Hygiene. Preventive Medicine
R Medicine > RM Therapeutics. Pharmacology
Divisions: Natural Sciences and Psychology
Publisher: BioMed Central
Date Deposited: 20 May 2019 09:51
Last Modified: 20 May 2019 10:00
DOI or Identification number: 10.1186/s13071-019-3490-z
URI: http://researchonline.ljmu.ac.uk/id/eprint/10727

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