Multiplex PCR for simultaneous genotyping of kdr mutations V410L, V1,016I and F1,534C inAedes aegypti(L.)

Abstract

BackgroundKnock down resistance (kdr) is the main mechanism that confers resistance to pyrethroids and DDT. This is a product of nonsynonymous mutations in the he voltage-gated sodium channel (VGSC) gene, these mutations produce a change of a single amino acid which reduces the affinity of the target site for the insecticide molecule. In Mexico, V410L, V1,016I and F1,534C mutations are common in pyrethroid-resistant Aedes aegypti (L.) populations.MethodsA multiplex PCR was developed to detect the V410L, V1,016I and F1,534C mutations in Ae. aegypti. The validation of the technique was carried out using wild populations previously characterized for the three mutations through allele-specific PCR (AS-PCR) and with different levels of genotypic frequencies.ResultsThe standardized protocol for multiplex endpoint PCR was highly effective in detecting 12 genotypes in five wild Ae. aegypti populations from Mexico. A complete concordance with AS-PCR was found for the simultaneous detection of the three kdr mutations.ConclusionsOur diagnostic method is highly effective for the simultaneous detection of V410L, V1,016I and F1,534C, when they co-occur. This technique represents a viable alternative to complement and strengthen current monitoring and resistance management strategies against Ae. aegypti.

Multiplex high resolution melting PCR for simultaneous genotyping of pyrethroid-resistance associated mutations in Aedes aegypti. First report on kdr mutations in wild populations from Argentina

“…For Ae. aegypti, the methods implemented to date are based either on allele-specific PCR [22], or in the use of TaqMan probes [15]. While the economic running cost of allele specific PCR is minor than TaqMan [23], it could be less reproducible by different laboratories, given the requirement of a careful setting of PCR conditions to avoid nonspecific amplifications.…”