Song Sishuo, Ji Fan, Zeng Qingdong, Ding Xiaoteng, Lin Jiahao, Wang Xinsheng, Xu Yuanhong, Deng Ruijie, Li Jinghong
College of Life Sciences, Institute of Biomedical Engineering, Qingdao University, Qingdao, Shandong 266071 China.
Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Tsinghua University, Beijing 100084, China.
Anal Chem. 2023 Mar 21;95(11):4966-4973. doi: 10.1021/acs.analchem.2c05115. Epub 2023 Mar 8.
Fungicide abuse leads to the emergence of fungicide-resistant fungal pathogens, thus posing a threat to agriculture and food safety. Here, we developed an isothermal amplification refractory mutation system (termed iARMS) allowing us to resolve genetic mutations, enabling rapid, sensitive, and potentially field-applicable detection of fungicide-resistant crop fungal pathogens. iARMS yielded a limit of detection of 25 aM via a cascade signal amplification strategy of recombinase polymerase amplification (RPA) and Cas12a-mediated collateral cleavage at 37 °C within 40 min. Specificity for fungicide-resistant () detection was guaranteed by RPA primers and the flexible sequence of gRNA. The iARMS assay allowed us to detect as low as 0.1% -mutated that showed resistance to the demethylase inhibitor (DMI), which was 50 times more sensitive than the sequencing techniques. Thus, it is promising for the discovery of rare fungicide-resistant isolates. We applied iARMS to investigate the emergence of fungicide-resistant in western China and found that its proportion was over 50% in Qinghai, Sichuan, and Xinjiang Province. iARMS can serve as a molecular diagnostic tool for crop diseases and facilitate precision plant disease management.
杀真菌剂的滥用导致了抗杀真菌剂的真菌病原体的出现，从而对农业和食品安全构成威胁。在此，我们开发了一种等温扩增难治性突变系统（称为iARMS），使我们能够解析基因突变，实现对农作物抗杀真菌剂真菌病原体的快速、灵敏且可能适用于田间的检测。iARMS通过重组酶聚合酶扩增（RPA）和Cas12a介导的旁切在37°C下40分钟内的级联信号放大策略，检测限达到25 aM。RPA引物和gRNA的灵活序列保证了对抗杀真菌剂（）检测的特异性。iARMS检测方法使我们能够检测到低至0.1%的对脱甲基酶抑制剂（DMI）具有抗性的突变，其灵敏度比测序技术高50倍。因此，它在发现罕见的抗杀真菌剂分离株方面很有前景。我们应用iARMS研究了中国西部抗杀真菌剂的出现情况，发现其在青海、四川和新疆的比例超过50%。iARMS可作为作物病害的分子诊断工具，促进精准植物病害管理。
