New and rapid visual detection assay for Trogoderma granarium Everts based on recombinase polymerase amplification and CRISPR/Cas12a.

Abstract

Khapra beetle (Trogoderma granarium Everts), one of the most important quarantine pests globally, is capable of causing severe infestation and huge economic loss to stored grain, and its interception rate has increased in major global trade countries over the past few years. However, difficulties remain in distinguishing this species with similar ones. In order to assist border ports and warehouses in khapra beetle's effective rapid identification as well as pest control at the early stages of monitoring or interception, we herein developed a new and rapid visual detection assay for T. granarium based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a system. We designed and selected the first khapra beetle-specific RPA primers and crRNA, and optimized the visualization reaction system (Cas12a/CrRNA=100 nM/500 nM). With only a 37°C-heat-source and a blue light torch, RPA and CRISPR/CAS12a-based visualization assays can be completed within 40 min to differentiate among khapra beetle and other nine similar Dermestidae species. After DNA extraction using a kit (4-5 h) or a simple method (5 min), the specific amplicons were obtained after a 15 min-RPA-reaction at 37°C, followed by a 15 min-color-reaction under 37°C in dark using CRISPR/CAS12a system and a fluorescent probe (5'-FAM/3'-BHQ1 labeled). This method is ingenious to low levels of DNA (10-1 ng/μL) and meets the sensitivity requirements for detecting a single khapra beetle's egg (about 0.7 mm). Our specificity and sensitivity analysis inferred that the present visualization system is effective to quickly and uniquely detect khapra beetle at room temperature (37°C), thereby preventing this species before they spread widely. Our study is suitable for being pushed forward in storage pest management and provides value as a reference for monitoring and identification of other pests. This article is protected by copyright. All rights reserved.