Multiplex PCR for simultaneous genotyping of kdr mutations V410L, V1016I and F1534C in Aedes aegypti (L.)

Abstract

Background: Knockdown resistance (kdr) is the main mechanism that confers resistance to pyrethroids and DDT. This is a product of non-synonymous mutations in the voltage-gated sodium channel (vgsc) gene, and 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, V1016I and F1534C mutations are common in pyrethroid-resistant Aedes aegypti (L.) populations. Methods: A multiplex PCR was developed to detect the V410L, V1016I and F1534C mutations in Ae. aegypti. The validation of the technique was carried out by DNA sequencing using field populations previously characterized for the three mutations through allele-specific PCR (AS-PCR) and with different levels of genotypic frequencies. Results: The standardized protocol for multiplex end-point PCR was highly effective in detecting 15 genotypes considering the three mutations V410L, V1106I and F1534C, in 12 field populations of Ae. aegypti from Mexico. A complete concordance with AS-PCR and DNA sequencing was found for the simultaneous detection of the three kdr mutations. Conclusions: Our diagnostic method is highly effective for the simultaneous detection of V410L, V1016I and F1534C, when they co-occur. This technique represents a viable alternative to complement and strengthen current monitoring and resistance management strategies against Ae. aegypti.

Impact of the V410L kdr mutation and co-occurring genotypes at kdr sites 1016 and 1534 in the VGSC on the probability of survival of the mosquito Aedes aegypti (L.) to Permanone in Harris County, TX, USA

“…Indeed, 81.8% of all females genotyped had at least one resistant allele at the 410 position ( Table 2 and Figs 1 and 2 ). These results match previous studies which had found high frequencies of the V410L genotype in combination with V1016I and F1534C genotypes throughout the world, including the Americas and Africa [ 20 , 26 – 28 , 45 – 47 ].…”

High DDT resistance without apparent association to kdr and Glutathione-S-transferase (GST) gene mutations in Aedes aegypti population at hotel compounds in Zanzibar

“…For the genotyping assay for polymorphisms V1016G/I, F1534C, and V410L, mosquito extracted DNA was investigated by AS-qPCR. Published primer mixes were used consisting of a common reverse primer and two specific primers targeting each polymorphic site [ 44 – 46 ]. The specificity of the primers was attained for the 3’-end.…”

“…Cycling conditions for V1016G consisted of a first denaturing step of 94°C for 10 min, then denaturing 94°C for 30 seconds, annealing time of 55°C for 30 sec, and elongation time of 72°C for 30 seconds for 40 cycles [ 45 ]. Cycling conditions for V410L consisted of a first denaturing step of 95°C for 10 min, then denaturing 95°C for 1 min, annealing time of 56°C for 20 sec, and elongation time of 72°C for 20 seconds for 40 cycles [ 46 ].…”

Deteksi Gen Kdr pada Nyamuk Anopheles di Kabupaten Maluku Tenggara Barat

“…Kajian resistensi vektor terhadap insektisida dengan teknik impregnated paper telah menunjukkan keberhasilan, 31,32 demikian juga dengan teknik molekuler, baik pada vektor malaria maupun DBD. [33][34][35][36] Sebab itu, pendekatan-pendekatan tersebut patut diaplikasikan dalam penentuan resistensi insektisida.…”