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A CRISPR/Cas12a-based portable platform for rapid detection of Leptosphaeria maculans in Brassica crops

Establishing a portable diagnostic method for identifying plant pathogens is essential to prevent the spread of plant disease, especially in field and customs inspections. Leptosphaeria maculans (L. maculans) is an aggressive fungus, which causes severe phoma stem canker of Brassica napus, responsib...

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Autores principales: Lei, Rong, Li, Yuan, Li, Limei, Wang, Jingyi, Cui, Zhenhai, Ju, Rui, Jiang, Li, Liao, Xiaoling, Wu, Pinshan, Wang, Xinyi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9493447/
https://www.ncbi.nlm.nih.gov/pubmed/36160953
http://dx.doi.org/10.3389/fpls.2022.976510
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author Lei, Rong
Li, Yuan
Li, Limei
Wang, Jingyi
Cui, Zhenhai
Ju, Rui
Jiang, Li
Liao, Xiaoling
Wu, Pinshan
Wang, Xinyi
author_facet Lei, Rong
Li, Yuan
Li, Limei
Wang, Jingyi
Cui, Zhenhai
Ju, Rui
Jiang, Li
Liao, Xiaoling
Wu, Pinshan
Wang, Xinyi
author_sort Lei, Rong
collection PubMed
description Establishing a portable diagnostic method for identifying plant pathogens is essential to prevent the spread of plant disease, especially in field and customs inspections. Leptosphaeria maculans (L. maculans) is an aggressive fungus, which causes severe phoma stem canker of Brassica napus, responsible for major yield losses of oilseed rape worldwide. In this study, CRISPR/Cas12a-based detection system and recombinase polymerase amplification (RPA) technique were employed to develop a rapid and sensitive detection method for identifying L. maculans. The involved RPA pre-amplification and CRISPR/Cas12a cleavage confer considerable sensitivity and selectivity, which can be finished within 45 min with a LOD of 4.7 genomic DNA copies. This detection system was further developed to two portable platforms, i.e., one-pot lateral flow detection and all-in-one chip lateral flow assay (AOCLFA), which integrates the lyophilized recombinase polymerase amplification (RPA) reagents and lyophilized Cas12a cleavage reagents in one tube or chip. The developed portable platforms have flexible portability and simple operation for the detection of L. maculans from plant tissues in the field. The proposed portable suitcase containing the minimum equipment, regents, and AOCLFA meets the practical needs of rapid on-site disease screening of plant fungi, port quarantine, or pathogen spreading control.
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spelling pubmed-94934472022-09-23 A CRISPR/Cas12a-based portable platform for rapid detection of Leptosphaeria maculans in Brassica crops Lei, Rong Li, Yuan Li, Limei Wang, Jingyi Cui, Zhenhai Ju, Rui Jiang, Li Liao, Xiaoling Wu, Pinshan Wang, Xinyi Front Plant Sci Plant Science Establishing a portable diagnostic method for identifying plant pathogens is essential to prevent the spread of plant disease, especially in field and customs inspections. Leptosphaeria maculans (L. maculans) is an aggressive fungus, which causes severe phoma stem canker of Brassica napus, responsible for major yield losses of oilseed rape worldwide. In this study, CRISPR/Cas12a-based detection system and recombinase polymerase amplification (RPA) technique were employed to develop a rapid and sensitive detection method for identifying L. maculans. The involved RPA pre-amplification and CRISPR/Cas12a cleavage confer considerable sensitivity and selectivity, which can be finished within 45 min with a LOD of 4.7 genomic DNA copies. This detection system was further developed to two portable platforms, i.e., one-pot lateral flow detection and all-in-one chip lateral flow assay (AOCLFA), which integrates the lyophilized recombinase polymerase amplification (RPA) reagents and lyophilized Cas12a cleavage reagents in one tube or chip. The developed portable platforms have flexible portability and simple operation for the detection of L. maculans from plant tissues in the field. The proposed portable suitcase containing the minimum equipment, regents, and AOCLFA meets the practical needs of rapid on-site disease screening of plant fungi, port quarantine, or pathogen spreading control. Frontiers Media S.A. 2022-09-08 /pmc/articles/PMC9493447/ /pubmed/36160953 http://dx.doi.org/10.3389/fpls.2022.976510 Text en Copyright © 2022 Lei, Li, Li, Wang, Cui, Ju, Jiang, Liao, Wu and Wang. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Lei, Rong
Li, Yuan
Li, Limei
Wang, Jingyi
Cui, Zhenhai
Ju, Rui
Jiang, Li
Liao, Xiaoling
Wu, Pinshan
Wang, Xinyi
A CRISPR/Cas12a-based portable platform for rapid detection of Leptosphaeria maculans in Brassica crops
title A CRISPR/Cas12a-based portable platform for rapid detection of Leptosphaeria maculans in Brassica crops
title_full A CRISPR/Cas12a-based portable platform for rapid detection of Leptosphaeria maculans in Brassica crops
title_fullStr A CRISPR/Cas12a-based portable platform for rapid detection of Leptosphaeria maculans in Brassica crops
title_full_unstemmed A CRISPR/Cas12a-based portable platform for rapid detection of Leptosphaeria maculans in Brassica crops
title_short A CRISPR/Cas12a-based portable platform for rapid detection of Leptosphaeria maculans in Brassica crops
title_sort crispr/cas12a-based portable platform for rapid detection of leptosphaeria maculans in brassica crops
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9493447/
https://www.ncbi.nlm.nih.gov/pubmed/36160953
http://dx.doi.org/10.3389/fpls.2022.976510
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