One-Pot Assay for Rapid Detection of Benzimidazole Resistance in Venturia carpophila by Combining RPA and CRISPR/Cas12a

文献类型： 外文期刊

第一作者： Hu, Jia-Jie

作者： Hu, Jia-Jie;Liu, Duo;Cai, Min-Zheng;Luo, Chao-Xi;Hu, Jia-Jie;Liu, Duo;Cai, Min-Zheng;Luo, Chao-Xi;Zhou, Yang;Yin, Wei-Xiao

作者机构：

关键词： fungicide resistance; one-pot detection; MBC fungicides; Venturia carpophila; RPA; CRISPR/Cas12a

期刊名称：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY （ 影响因子：6.1； 五年影响因子：6.3 ）

ISSN： 0021-8561

年卷期： 2023 年 71 卷 3 期

页码：

收录情况： SCI

摘要： High resistance to benzimidazole fungicides in Venturia carpophila is caused by the point mutation E198K of the beta-tubulin (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 x 10(3) fg mu L-1 V. carpophila genomic DNA in 45 min at 37 degrees 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.

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