High resistance to benzimidazole fungicides in Venturia carpophila is caused by the point mutation E198K of the βtubulin (TUB2) gene. Traditional methods for detection of fungicide resistance are time-consuming, which are routinely based on tedious operation, reliance on expensive equipment, and specially trained people. Therefore, it is important to establish efficient methods for field detection of benzimidazole resistance in V. carpophila to make suitable management strategies and ensure food safety. Based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a, a rapid one-pot assay ORCas12a-BRVc (one-pot RPA-CRISPR/Cas12 platform) was established for the detection of benzimidazole resistance in V. carpophila. The ORCas12a-BRVc assay enabled one-pot detection by adding components at the bottom and wall of the tube separately, solving the problems of aerosol contamination and decreased sensitivity caused by competing DNA substrates between Cas12a cleavage and RPA amplification. The ORCas12a-BRVc assay could accomplish the detection with a minimum of 7.82 × 10 3 fg μL −1 V. carpophila genomic DNA in 45 min at 37 °C. Meanwhile, this assay showed excellent specificity due to the specific recognition ability of the Cas12a-crRNA complex. Further, we combined a method that could rapidly extract DNA from V. carpophila within 2 min with the ORCas12a-BRVc to achieve more rapid and simple detection of V. carpophila with benzimidazole resistance in fields. The ORCas12a-BRVc assay has the advantages of simplicity, rapidity, high sensitivity, high specificity, and ease of operation without the need for precision instruments and the need to isolate and culture pathogens. This assay is the first application of the one-pot platform based on the combination of RPA and CRISPR/Cas12a in fungicide resistance detection and can be used for monitoring of resistant populations in fields, providing guidance on making suitable management strategies for peach scab.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.