Development of a multiplex SNaPshot assay for simultaneous detection of multiple insecticide-resistance mutations in Anopheles sinensis

Background Anopheles sinensis , the primary malaria vector in China, is a potential threat to the prevention of re-establishment of malaria transmission following imported cases. The extensive use of insecticides has led to widespread resistance. Conventional bioassays and molecular detection are limited in sensitivity, throughput, and efficiency, underscoring the need for a rapid and cost-effective genotyping tool for large-scale multiple insecticide-resistance markers surveillance. Methods A multiplex SNaPshot assay was developed to simultaneously detect six SNPs ( kdr1014-F , kdr1014-R , Ace-1 , RDL296 , RDL327 , and RDL345 ) in An. sinensis . Positive plasmids carrying known resistance-associated genotypes and laboratory-reared An. sinensis samples were used to optimize and validate the reactions. Wild An. sinensis collected from Henan Puyang (HN-PY) and Anhui Shucheng (AH-SC) were used for bioassays with 0.05% deltamethrin, 0.15% beta-cyfluthrin, 5% malathion, 0.1% propoxur, and 0.06% flufenoxuron. Then samples randomly selected on the basis of 24 h survival status were genotyped via PCR–Sanger sequencing and the SNaPshot assay. Consistency was assessed using the Kappa testing, and genotype–phenotype correlations were evaluated by chi-squared tests. Results Bioassays showed that the HN-PY An. sinensis population was resistant to deltamethrin, beta-cyfluthrin, malathion, propoxur, and flufenoxuron, with adjusted mortality rates of 5.2%, 24.4%, 9.1%, 24.3%, and 5.8%, respectively. In contrast, the AH-SC population remained susceptible to malathion (100% mortality) but exhibited resistance to deltamethrin, beta-cyfluthrin, propoxur, and flufenoxuron, with adjusted mortality rates of 71.6%, 78.6%, 44.9%, and 33.4%, respectively. Multiplex SNaPshot genotyping of 272 field-collected mosquitoes revealed high frequencies of resistance-associated mutations at the kdr 1014-F, kdr 1014-R, Ace-1 , RDL 296, RDL 327, and RDL 345 loci, with frequencies of 4.7%, 99.3%, 53.3%, 89.7%, 96.0%, and 53.3% in the HN-PY population and 25.5%, 78.0%, 55.3%, 89.8%, 89.8%, and 46.3% in the AH-SC population, respectively. Additionally, the SNaPshot assay showed strong agreement with PCR–Sanger sequencing (Kappa > 0.90 at all loci). Conclusions The multiplex SNaPshot assay provides a reliable, rapid, and economical tool for detecting multiple resistance mutations in An. sinensis . Its application in routine entomological surveillance will provide an alternative tool for early warning of insecticide resistance.