New and rapid visual detection assay for Hyphantria cunea Drury based on recombinase polymerase amplification and CRISPR/Cas12a
Hyphantria cunea Drury is one of the most important forestry pests in China,with numerous host plants and wide distribution ranges.Based on recombinant polymerase amplification(RPA)and CRISPR detection techniques,we constructed the RPA-CRISPR/Cas12a Molecular Detection System of H.cunea.Specific RPA primers and crRNA were designed based on the CO I gene fragment data from H.cunea and its closely related and similar species in the NCBI database,as well as the sequencing results of insect samples.An RPA-CRISPR/Cas12a molecular detection system was constructed,through RPA of target sequences and the reaction conditions were optimized with CRISPR detection technology.The system demonstrates a sensitivity of up to 1.0×10-1 ng/µL,a hundredfold improvement over traditional agarose gel electrophoresis methods.The test can be completed in 30 minutes.Our study provides theoretical and practical support for visual detection of H.cunea,contributing to the protection of forest resources and ecological environments.
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