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A multiplex PCR for detection of knockdown resistance mutations, V1016G and F1534C, in pyrethroid-resistant Aedes aegypti.

Abstract

BACKGROUND

Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism of the dengue vector Aedes aegypti mosquitoes against pyrethroids. In many countries in Asia, a valine to glycine substitution (V1016G) and a phenylalanine to cysteine substitution (F1534C) are common in Ae. aegypti populations. The G1016 and C1534 allele frequencies have been increasing in recent years, and hence there is a need to have a simple and inexpensive tool to monitor the alleles in large scale.

METHODS

A multiplex PCR to detect V1016G and F1534C mutations has been developed in the current study. This study utilized primers from previous studies for detecting the mutation at position 1016 and newly designed primers to detect variants at position 1534. The PCR conditions were validated and compared with DNA sequencing using known kdr mutant laboratory strains and field collected mosquitoes. The efficacy of this method was also compared with allele-specific PCR (AS-PCR).

RESULTS

The results of our multiplex PCR were in complete agreement with sequencing data and better than the AS-PCR. In addition, the efficiency of two non-toxic DNA staining dyes, Ultrapower™ and RedSafe™, were evaluated by comparing with ethidium bromide (EtBr) and the results were satisfactory.

CONCLUSIONS

Our multiplex PCR method is highly reliable and useful for implementing vector surveillance in locations where the two alleles co-occur.

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  • Authors+Show Affiliations

    ,

    Graduate School, Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.

    ,

    Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.

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    Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.

    ,

    Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand.

    ,

    School of Earth and Environmental Sciences, Faculty of Science and Engineering, University of Manchester, Manchester, UK.

    Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. pradya.somboon@cmu.ac.th.

    Source

    Parasites & vectors 10:1 2017 Oct 10 pg 465

    Pub Type(s)

    Journal Article

    Language

    eng

    PubMed ID

    29017613