Development of PCR and RPA-LFD molecular diagnostic assays for monitoring pyrethroid resistance in Chinese populations of Spodoptera exigua based on an intronic indel marker.

Widespread lambda-cyhalothrin resistance in Spodoptera exigua severely limits chemical control efficacy. Here, we assessed the frequency of resistance-associated CYP9A9a mutations across wild populations. Three mutations (V40L, Q160H and T208A) were highly prevalent in all six field populations tested (carrier frequencies: 70.8%-100%) but absent in the susceptible WH-S strain. Genetic analysis confirmed that a 55-bp intronic insertion in the resistant CYP9A9a allele is tightly linked to the resistant phenotype. Targeting this insertion, we developed a PCR genotyping assay that achieved 90.91%-100% sensitivity compared with Sanger sequencing across six field populations. Additionally, we developed a rapid recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay targeting the same 55-bp insertion. Under optimized conditions (37 °C for 15 min), the RPA-LFD assay was highly specific to CYP9A9a resistant allele of S. exigua, showing no cross-reactivity with susceptible or blank controls, with a detection limit of 1 pg/μL. Together, these PCR and RPA-LFD diagnostic systems offer practical complementary molecular tools for high-throughput laboratory screening and rapid field-based surveillance of pyrethroid resistance alleles in S. exigua.