Department of Entomology, College of Plant Protection, China Agricultural University, Beijing 100193, China.
Department of Plant Biosecurity and MOA Key Laboratory of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing 100193, China.
Sci Total Environ. 2024 Nov 15;951:175712. doi: 10.1016/j.scitotenv.2024.175712. Epub 2024 Aug 22.
Insecticide resistance monitoring is essential for guiding chemical pest control and resistance management policies. Currently, rapid and effective technology for monitoring the resistance of tiny insects in the field is absent. Aphis gossypii Glover is a typical tiny insect, and one of the most frequently reported insecticide-resistant pests. In this study, we established a novel CRISPR/Cas12a-based rapid visual detection approach for detecting the V62I and R81T mutations in the β1 subunit of the nAChR in A. gossypii, to reflect target-site resistance to imidacloprid. Based on the nAChR β1 subunit gene in A. gossypii, the V62I/R81T-specific RPA primers and crRNAs were designed, and the ratio of 10 μM/2 μM/10 μM for ssDNA/Cas12a/crRNA was selected as the optimal dosage for the CRISPR reaction, ensuring that Cas12a only accurately recognizes imidacloprid-resistance templates. Our data show that the field populations of resistant insects possessing V62I and R81T mutations to imidacloprid can be accurately identified within one hour using the RPA-CRISPR/Cas12a detection approach under visible blue light at 440-460 nm. The protocol for RPA-CRISPR detection necessitates a single less than 2 mm specimen of A. gossypii tissues to perform RPA-CRISPR detection, and the process only requires a container at 37 °C and a portable blue light at 440-460 nm. Our research represents the first application of RPA-CRISPR technology in insecticide resistance detection, offers a new method for the resistance monitoring of A. gossypii or other tiny insects, helps delay the development of resistance to imidacloprid, improves the sustainability of chemical control, and provides theoretical guidance for managing pest resistance.
昆虫抗药性监测对于指导化学防治和抗药性管理策略至关重要。目前，缺乏快速有效的田间微小昆虫抗药性监测技术。棉蚜是一种典型的微小昆虫，也是报告最多的抗药性害虫之一。在本研究中，我们建立了一种新的基于 CRISPR/Cas12a 的快速视觉检测方法，用于检测棉蚜烟碱型乙酰胆碱受体β1 亚基中的 V62I 和 R81T 突变，以反映对吡虫啉的靶标部位抗性。基于棉蚜烟碱型乙酰胆碱受体β1 亚基基因，设计了 V62I/R81T 特异性 RPA 引物和 crRNA，选择 10 μM/2 μM/10 μM 的 ssDNA/Cas12a/crRNA 比例作为 CRISPR 反应的最佳剂量，确保 Cas12a 只能准确识别吡虫啉抗性模板。我们的数据表明，使用 RPA-CRISPR/Cas12a 检测方法，在 440-460nm 可见蓝光下，仅需 1 小时即可准确识别携带 V62I 和 R81T 突变的抗药性田间种群。RPA-CRISPR 检测的方案需要不到 2mm 的棉蚜组织的单一样本即可进行 RPA-CRISPR 检测，该过程仅需要一个 37°C 的容器和一个便携式 440-460nm 的蓝光。我们的研究代表了 RPA-CRISPR 技术在杀虫剂抗性检测中的首次应用，为棉蚜或其他微小昆虫的抗性监测提供了一种新方法，有助于延缓对吡虫啉的抗性发展，提高化学防治的可持续性，并为管理害虫抗性提供理论指导。
