开发一种 RPA-CRISPR/Cas12a 检测方法,用于快速灵敏地诊断植物检疫真菌 Setophoma terrestris。

IF 4.2 2区 生物学 Q2 MICROBIOLOGY Journal of Fungi Pub Date : 2024-10-15 DOI:10.3390/jof10100716
Peng Zhao, Zhipeng Feng, Lei Cai, Dorji Phurbu, Weijun Duan, Fuhong Xie, Xuelian Li, Fang Liu
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引用次数: 0

摘要

Setophoma terrestris 是一种重要的植物病原真菌,中国已将其列为受植物检疫进口管制的有害真菌。这种病原菌对多种植物构成威胁,尤其是作为洋葱粉红根腐病的病原菌,它是洋葱最严重的病害之一。为了对 S. terrestris 进行快速鉴定和预警,防止其扩散,我们利用重组酶聚合酶扩增(RPA)技术,结合 CRISPR/Cas12a 裂解和基于荧光的检测系统或纸质侧流条带,开发了一种快速、准确和直观的病原体诊断方法。所开发的 RPA-CRISPR/Cas12a 检测方法在检测赤潮沙雷氏菌方面具有显著的特异性。此外,该方案检测病原体的灵敏度为 0.01 pg/μL,明显优于现有的基于 qPCR 的检测方法 1 pg/μL 的灵敏度。整个诊断过程,包括 DNA 提取、RPA 反应、Cas12a 裂解和结果判读,可在 40 分钟内完成。此外,该检测方法在受感染植物样本中的成功应用凸显了其在农业环境中快速准确检测病原体的潜力。总之,这种 RPA-CRISPR/Cas12a 诊断方法为检疫和疾病管理提供了一种有潜在价值的技术解决方案。
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Development of an RPA-CRISPR/Cas12a Assay for Rapid and Sensitive Diagnosis of Plant Quarantine Fungus Setophoma terrestris.

Setophoma terrestris is an important phytopathogenic fungus listed by China as a harmful fungus subject to phytosanitary import control. This pathogen is a threat to a wide range of plants, particularly as the causal agent of onion pink root rot, one of the most severe diseases of onions. In order to provide rapid identification and early warning of S. terrestris and prevent its spread, we have developed a rapid, accurate, and visually intuitive diagnostic assay for this pathogen, by utilizing recombinase polymerase amplification (RPA), coupled with CRISPR/Cas12a cleavage and fluorescence-based detection systems or paper-based lateral flow strips. The developed RPA-CRISPR/Cas12a assay exhibited remarkable specificity for the detection of S. terrestris. Moreover, this protocol can detect the pathogen at a sensitivity level of 0.01 pg/μL, which significantly outperforms the 1 pg/μL sensitivity achieved by the existing qPCR-based detection method. The entire diagnostic procedure, including DNA extraction, the RPA reaction, the Cas12a cleavage, and the result interpretation, can be accomplished in 40 min. Furthermore, the successful application of the assay in infected plant samples highlighted its potential for rapid and accurate pathogen detection in agricultural settings. In summary, this RPA-CRISPR/Cas12a diagnostic method offers a potentially valuable technological solution for quarantine and disease management.

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来源期刊
Journal of Fungi
Journal of Fungi Medicine-Microbiology (medical)
CiteScore
6.70
自引率
14.90%
发文量
1151
审稿时长
11 weeks
期刊介绍: Journal of Fungi (ISSN 2309-608X) is an international, peer-reviewed scientific open access journal that provides an advanced forum for studies related to pathogenic fungi, fungal biology, and all other aspects of fungal research. The journal publishes reviews, regular research papers, and communications in quarterly issues. Our aim is to encourage scientists to publish their experimental and theoretical results in as much detail as possible. Therefore, there is no restriction on paper length. Full experimental details must be provided so that the results can be reproduced.
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