Peng Zhao, Zhipeng Feng, Lei Cai, Dorji Phurbu, Weijun Duan, Fuhong Xie, Xuelian Li, Fang Liu
{"title":"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。","authors":"Peng Zhao, Zhipeng Feng, Lei Cai, Dorji Phurbu, Weijun Duan, Fuhong Xie, Xuelian Li, Fang Liu","doi":"10.3390/jof10100716","DOIUrl":null,"url":null,"abstract":"<p><p><i>Setophoma terrestris</i> 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 <i>S. terrestris</i> 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 <i>S. terrestris.</i> 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.</p>","PeriodicalId":15878,"journal":{"name":"Journal of Fungi","volume":"10 10","pages":""},"PeriodicalIF":4.2000,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11509020/pdf/","citationCount":"0","resultStr":"{\"title\":\"Development of an RPA-CRISPR/Cas12a Assay for Rapid and Sensitive Diagnosis of Plant Quarantine Fungus <i>Setophoma terrestris</i>.\",\"authors\":\"Peng Zhao, Zhipeng Feng, Lei Cai, Dorji Phurbu, Weijun Duan, Fuhong Xie, Xuelian Li, Fang Liu\",\"doi\":\"10.3390/jof10100716\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p><i>Setophoma terrestris</i> 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 <i>S. terrestris</i> 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 <i>S. terrestris.</i> 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.</p>\",\"PeriodicalId\":15878,\"journal\":{\"name\":\"Journal of Fungi\",\"volume\":\"10 10\",\"pages\":\"\"},\"PeriodicalIF\":4.2000,\"publicationDate\":\"2024-10-15\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11509020/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Fungi\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.3390/jof10100716\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Fungi","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.3390/jof10100716","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MICROBIOLOGY","Score":null,"Total":0}
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.
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.