Development and application of a PCR–RFLP assay revealing widespread distribution of the pyrethroid resistance-associated VGSC V1016G mutation in Aedes albopictus from Guangyuan City, Sichuan Province of China

Background Aedes albopictus is a primary vector of multiple arboviruses, including dengue, chikungunya, yellow fever, and Zika virus. Its control relies heavily on pyrethroid insecticides. The V1016G mutation in the voltage-gated sodium channel (VGSC) is a well-documented mechanism conferring pyrethroid resistance in Ae. albopictus , which directly challenges the efficacy of pyrethroid-based control. Understanding of the status of insecticide resistance will offer insights to inform evidence-based vector management. However, current phenotypic monitoring is laborious and time-consuming, highlighting the need for rapid and reliable genotyping tools. Methods To detect the V1016G mutation, we developed a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assay. This assay was then applied to genotype 208 field-collected Ae. albopictus mosquitoes. These samples were collected in 2024 from seven counties/districts within Guangyuan City, a prefecture in northern Sichuan, China. Results The PCR–RFLP assay demonstrated 100% concordance with Sanger sequencing results. Genotyping confirmed the widespread presence of the 1016G allele, with frequencies ranging from 3.13% to 14.06%. The resistance allele (1016G) was exclusively detected in heterozygotes, and all populations conformed to Hardy–Weinberg equilibrium ( P > 0.05). Furthermore, no significant temporal changes in allele frequencies were detected between 2020 and 2024 across the populations ( P > 0.05). Conclusions This study established a cost-effective and reliable PCR–RFLP assay for detecting the V1016G mutation in Ae. albopictus , and demonstrated the widespread distribution of this mutation across Guangyuan City, Sichuan Province of China.