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Updated: Jun 28, 2026
A Multi-detection Assay for Malaria Transmitting Mosquitoes
Published on: February 28, 2015
Development of a multiplex SNaPshot assay for simultaneous detection of multiple insecticide-resistance mutations in
Yi Xin1, Zhenyu Yue1, Shuning Yan2
1National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; Chinese Center for Tropical Diseases Research; National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases; WHO Collaborating Centre for Tropical Diseases; Key Laboratory on Parasite and Vector Biology, Ministry of Health; WHO Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Ministry of Science and Technology, Shanghai, 200025, China.
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 kdr1014-F, kdr1014-R, Ace-1, RDL296, RDL327, and RDL345 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.
