Rapid and accurate detection of Fusarium oxysporum f. sp. Lycopersici using one-pot, one-step LAMP-CRISPR/Cas12b method
IntroductionFusarium oxysporum f. sp. Lycopersici (Fol) is one of the most devastating plant pathogenic fungi, the causal agent of root rot for Atractylides macrocephala Koidz (AMK). An accurate rapid and convenient diagnosis for FoL detection is essential for determining management practices and pr...
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Frontiers Media S.A.
2024-12-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2024.1485884/full |
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| author | Shijie Dai Yangsheng Wu Na Zhu Yujin Zhao Mingjiang Mao Zheming Li Bo Zhu Weichun Zhao Xiaofeng Yuan Xiaofeng Yuan |
| author_facet | Shijie Dai Yangsheng Wu Na Zhu Yujin Zhao Mingjiang Mao Zheming Li Bo Zhu Weichun Zhao Xiaofeng Yuan Xiaofeng Yuan |
| author_sort | Shijie Dai |
| collection | DOAJ |
| description | IntroductionFusarium oxysporum f. sp. Lycopersici (Fol) is one of the most devastating plant pathogenic fungi, the causal agent of root rot for Atractylides macrocephala Koidz (AMK). An accurate rapid and convenient diagnosis for FoL detection is essential for determining management practices and preventing future losses for AMK.MethodsHere, we developed a novel method for Fol detection by integrating loop-mediated isothermal amplification (LAMP) assay and CRISPR/Cas12b detection in one-pot, and the whole reaction can simultaneously amplify and detect the target gene of Fol in one-step.ResultsThe total time of the present method is limited to 45 min and isothermally performed at 60°C. The limit of detection of this assay is 88.9 copies per reaction. The specificity of the LAMP-CRISPR/Cas12b method was 100% without any cross-reaction of other pathogens. A total of 24 nucleic acid samples were used to evaluate the performance of the LAMP-CRISPR/Cas12b method, including 12 with-Fol and 12 without-Fol. Compared with the gold standard results from real-time PCR, the present method provides a sensitivity of 100% (12/12), specificity of 100% (12/12), and consistency of 100% (24/24).DiscussionTogether, our preliminary results illustrated that the LAMP-CRISPR/Cas12b method is a rapid simple, and reliable tool for Fol diagnosis and could be applied in point-of-need phytopathogen detection. |
| format | Article |
| id | doaj-art-5207e30f0e0e4e06966feea4216ea2d2 |
| institution | DOAJ |
| issn | 1664-462X |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj-art-5207e30f0e0e4e06966feea4216ea2d22025-08-20T02:52:12ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2024-12-011510.3389/fpls.2024.14858841485884Rapid and accurate detection of Fusarium oxysporum f. sp. Lycopersici using one-pot, one-step LAMP-CRISPR/Cas12b methodShijie Dai0Yangsheng Wu1Na Zhu2Yujin Zhao3Mingjiang Mao4Zheming Li5Bo Zhu6Weichun Zhao7Xiaofeng Yuan8Xiaofeng Yuan9College of Life Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, ChinaCollege of Life Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, ChinaCollege of Life Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, ChinaCollege of Life Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, ChinaCollege of Life Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, ChinaCollege of Life Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, ChinaSchool of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, ChinaCollege of Life Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, ChinaCollege of Life Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, ChinaAcademy of Chinese Medical Sciences, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, ChinaIntroductionFusarium oxysporum f. sp. Lycopersici (Fol) is one of the most devastating plant pathogenic fungi, the causal agent of root rot for Atractylides macrocephala Koidz (AMK). An accurate rapid and convenient diagnosis for FoL detection is essential for determining management practices and preventing future losses for AMK.MethodsHere, we developed a novel method for Fol detection by integrating loop-mediated isothermal amplification (LAMP) assay and CRISPR/Cas12b detection in one-pot, and the whole reaction can simultaneously amplify and detect the target gene of Fol in one-step.ResultsThe total time of the present method is limited to 45 min and isothermally performed at 60°C. The limit of detection of this assay is 88.9 copies per reaction. The specificity of the LAMP-CRISPR/Cas12b method was 100% without any cross-reaction of other pathogens. A total of 24 nucleic acid samples were used to evaluate the performance of the LAMP-CRISPR/Cas12b method, including 12 with-Fol and 12 without-Fol. Compared with the gold standard results from real-time PCR, the present method provides a sensitivity of 100% (12/12), specificity of 100% (12/12), and consistency of 100% (24/24).DiscussionTogether, our preliminary results illustrated that the LAMP-CRISPR/Cas12b method is a rapid simple, and reliable tool for Fol diagnosis and could be applied in point-of-need phytopathogen detection.https://www.frontiersin.org/articles/10.3389/fpls.2024.1485884/fullFOLLAMPCas12bone-potpoint-of-needphytopathogen detection |
| spellingShingle | Shijie Dai Yangsheng Wu Na Zhu Yujin Zhao Mingjiang Mao Zheming Li Bo Zhu Weichun Zhao Xiaofeng Yuan Xiaofeng Yuan Rapid and accurate detection of Fusarium oxysporum f. sp. Lycopersici using one-pot, one-step LAMP-CRISPR/Cas12b method Frontiers in Plant Science FOL LAMP Cas12b one-pot point-of-need phytopathogen detection |
| title | Rapid and accurate detection of Fusarium oxysporum f. sp. Lycopersici using one-pot, one-step LAMP-CRISPR/Cas12b method |
| title_full | Rapid and accurate detection of Fusarium oxysporum f. sp. Lycopersici using one-pot, one-step LAMP-CRISPR/Cas12b method |
| title_fullStr | Rapid and accurate detection of Fusarium oxysporum f. sp. Lycopersici using one-pot, one-step LAMP-CRISPR/Cas12b method |
| title_full_unstemmed | Rapid and accurate detection of Fusarium oxysporum f. sp. Lycopersici using one-pot, one-step LAMP-CRISPR/Cas12b method |
| title_short | Rapid and accurate detection of Fusarium oxysporum f. sp. Lycopersici using one-pot, one-step LAMP-CRISPR/Cas12b method |
| title_sort | rapid and accurate detection of fusarium oxysporum f sp lycopersici using one pot one step lamp crispr cas12b method |
| topic | FOL LAMP Cas12b one-pot point-of-need phytopathogen detection |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2024.1485884/full |
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