A tiny sample rapid visual detection technology for imidacloprid resistance in Aphis gossypii by CRISPR/Cas12a.

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.