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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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      A multiplex SNaPshot assay was developed to simultaneously detect six SNPs (\n                      <jats:italic>kdr1014-F<\/jats:italic>\n                      ,\n                      <jats:italic>kdr1014-R<\/jats:italic>\n                      ,\n                      <jats:italic>Ace-1<\/jats:italic>\n                      ,\n                      <jats:italic>RDL296<\/jats:italic>\n                      ,\n                      <jats:italic>RDL327<\/jats:italic>\n                      , and\n                      <jats:italic>RDL345<\/jats:italic>\n                      ) in\n                      <jats:italic>An. sinensis<\/jats:italic>\n                      . Positive plasmids carrying known resistance-associated genotypes and laboratory-reared\n                      <jats:italic>An. sinensis<\/jats:italic>\n                      samples were used to optimize and validate the reactions. Wild\n                      <jats:italic>An. sinensis<\/jats:italic>\n                      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\u00a0h survival status were genotyped via PCR\u2013Sanger sequencing and the SNaPshot assay. Consistency was assessed using the Kappa testing, and genotype\u2013phenotype correlations were evaluated by chi-squared tests.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Bioassays showed that the HN-PY\n                      <jats:italic>An. sinensis<\/jats:italic>\n                      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\n                      <jats:italic>kdr<\/jats:italic>\n                      1014-F,\n                      <jats:italic>kdr<\/jats:italic>\n                      1014-R,\n                      <jats:italic>Ace-1<\/jats:italic>\n                      ,\n                      <jats:italic>RDL<\/jats:italic>\n                      296,\n                      <jats:italic>RDL<\/jats:italic>\n                      327, and\n                      <jats:italic>RDL<\/jats:italic>\n                      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\u2013Sanger sequencing (Kappa\u2009&gt;\u20090.90 at all loci).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      The multiplex SNaPshot assay provides a reliable, rapid, and economical tool for detecting multiple resistance mutations in\n                      <jats:italic>An. sinensis<\/jats:italic>\n                      . Its application in routine entomological surveillance will provide an alternative tool for early warning of insecticide resistance.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Graphical Abstract<\/jats:title>\n                  <\/jats:sec>","DOI":"10.1186\/s13071-026-07250-2","type":"journal-article","created":{"date-parts":[[2026,2,19]],"date-time":"2026-02-19T11:16:39Z","timestamp":1771499799000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a multiplex SNaPshot assay for simultaneous detection of multiple insecticide-resistance mutations in Anopheles sinensis"],"prefix":"10.1186","volume":"19","author":[{"given":"Yi","family":"Xin","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhenyu","family":"Yue","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shuning","family":"Yan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chenghang","family":"Yu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mowen","family":"Liu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shuo","family":"Yang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ruili","family":"Xie","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bin","family":"Zheng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jianhai","family":"Yin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bin","family":"Xu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2026,2,19]]},"reference":[{"key":"7250_CR1","volume-title":"Vector-borne diseases","author":"World Health Organization","year":"2024","unstructured":"World Health Organization. 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