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

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. Electronic supplementary material The online version of this article (10.1186/s13071-019-3490-z) contains supplementary material, which is available to authorized users.

Detection of the Nav channel kdr-like mutation and modeling of factors affecting survivorship of Culex quinquefasciatus mosquitoes from six areas of Harris County (Houston), Texas, after permethrin field-cage tests

“…However, the primers published by Chen et al (2010) are 19-23 bp, while the primers in this study are longer, 24-29 bp, for increasing specificity [27]. Martins et al (2019) developed an engineered-tail allele-specific-PCR (ETAS-PCR) for detecting L1014F in Cx. quinquefasciatus [28].…”

“…Martins et al (2019) developed an engineered-tail allele-specific-PCR (ETAS-PCR) for detecting L1014F in Cx. quinquefasciatus [28]. ETAS-PCR diagnostics relies on the design of different tail sequences incorporating an enzyme cutting site, each associated with a specific kdr-like mutations.…”

Assessment of susceptible Culex quinquefasciatus larvae in Indonesia to different insecticides through metabolic enzymes and the histopathological midgut

“…Currently, many reports have shown that the larval and adult stages of Cx. quinquefasciatus sre resistant to synthetic insecticides ( Lopes et al., 2019 ; Atyame et al., 2019 ; Nachaiwieng et al., 2021 ) because of knockdown resistance ( kdr) gene mutations, such as L1014F, G1195S ace-1, and the cytochrome P450 genes CYP9J45, CYP9J40, and CYP6AA7 ( Delannay et al., 2018 ; Silva Martins et al., 2019 ; Maestre-Serrano et al., 2020 ; Yoshimizu et al., 2020 ). These kdr gene mutations of Cx.…”

Establishment of an innovative and sustainable PCR technique for 1534 locus mutation of the knockdown resistance (kdr) gene in the dengue vector Aedes albopictus

“…Consequently, more cost-effective and rapid single-tube assays, e.g. multiplex PCR and tri-allelic PCR [18, 29], should be taken into account urgently. In general, the degree of resistance and the frequency of susceptible alleles detected by AS-PCR were almost the same as those by sequencing, meaning AS-PCR can accurately reflect the resistance levels of a given Ae.…”