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Reverse transcription-recombinase-aided amplification and CRISPR/Cas12a-based visual detection of maize chlorotic mottle virus
Maize chlorotic mottle virus (MCMV) is one of the important quarantine pathogens in China. It often co-infects with one or two viruses in the family Potyviridae and causes maize lethal necrosis disease. Therefore, an accurate and sensitive method for the detection of MCMV is urgently needed. Combine...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9207886/ https://www.ncbi.nlm.nih.gov/pubmed/35757182 http://dx.doi.org/10.1186/s42483-022-00128-y |
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author | Duan, Xueyan Ma, Wendi Jiao, Zhiyuan Tian, Yiying Ismail, Ragab Gomaa Zhou, Tao Fan, Zaifeng |
author_facet | Duan, Xueyan Ma, Wendi Jiao, Zhiyuan Tian, Yiying Ismail, Ragab Gomaa Zhou, Tao Fan, Zaifeng |
author_sort | Duan, Xueyan |
collection | PubMed |
description | Maize chlorotic mottle virus (MCMV) is one of the important quarantine pathogens in China. It often co-infects with one or two viruses in the family Potyviridae and causes maize lethal necrosis disease. Therefore, an accurate and sensitive method for the detection of MCMV is urgently needed. Combined with reverse transcription and recombinase-aided amplification, we developed a CRISPR/Cas12a-based visual nucleic acid detection system targeting the MCMV coat protein gene. The whole process can be completed within 45 min with high sensitivity. This system could detect cDNAs diluted up to 10(–5) when 2000 ng of total RNA was used for reverse transcription. The Cas12a/crRNA complex designed for MCMV detection could recognize and cleave the targeted double-stranded DNA, and ultimately cleave the single-stranded DNA probes and produce fluorescent signals. The green fluorescence produced under blue light (440–460 nm) in this procedure could be observed by the naked eye. Since this novel method is specific, rapid, sensitive and does not require special instruments and technical expertise, it should be suitable for on-site visual detection of MCMV in seeds, plants of maize and potentially in its insect vectors. |
format | Online Article Text |
id | pubmed-9207886 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-92078862022-06-21 Reverse transcription-recombinase-aided amplification and CRISPR/Cas12a-based visual detection of maize chlorotic mottle virus Duan, Xueyan Ma, Wendi Jiao, Zhiyuan Tian, Yiying Ismail, Ragab Gomaa Zhou, Tao Fan, Zaifeng Phytopathol Res Research Maize chlorotic mottle virus (MCMV) is one of the important quarantine pathogens in China. It often co-infects with one or two viruses in the family Potyviridae and causes maize lethal necrosis disease. Therefore, an accurate and sensitive method for the detection of MCMV is urgently needed. Combined with reverse transcription and recombinase-aided amplification, we developed a CRISPR/Cas12a-based visual nucleic acid detection system targeting the MCMV coat protein gene. The whole process can be completed within 45 min with high sensitivity. This system could detect cDNAs diluted up to 10(–5) when 2000 ng of total RNA was used for reverse transcription. The Cas12a/crRNA complex designed for MCMV detection could recognize and cleave the targeted double-stranded DNA, and ultimately cleave the single-stranded DNA probes and produce fluorescent signals. The green fluorescence produced under blue light (440–460 nm) in this procedure could be observed by the naked eye. Since this novel method is specific, rapid, sensitive and does not require special instruments and technical expertise, it should be suitable for on-site visual detection of MCMV in seeds, plants of maize and potentially in its insect vectors. BioMed Central 2022-06-20 2022 /pmc/articles/PMC9207886/ /pubmed/35757182 http://dx.doi.org/10.1186/s42483-022-00128-y Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Research Duan, Xueyan Ma, Wendi Jiao, Zhiyuan Tian, Yiying Ismail, Ragab Gomaa Zhou, Tao Fan, Zaifeng Reverse transcription-recombinase-aided amplification and CRISPR/Cas12a-based visual detection of maize chlorotic mottle virus |
title | Reverse transcription-recombinase-aided amplification and CRISPR/Cas12a-based visual detection of maize chlorotic mottle virus |
title_full | Reverse transcription-recombinase-aided amplification and CRISPR/Cas12a-based visual detection of maize chlorotic mottle virus |
title_fullStr | Reverse transcription-recombinase-aided amplification and CRISPR/Cas12a-based visual detection of maize chlorotic mottle virus |
title_full_unstemmed | Reverse transcription-recombinase-aided amplification and CRISPR/Cas12a-based visual detection of maize chlorotic mottle virus |
title_short | Reverse transcription-recombinase-aided amplification and CRISPR/Cas12a-based visual detection of maize chlorotic mottle virus |
title_sort | reverse transcription-recombinase-aided amplification and crispr/cas12a-based visual detection of maize chlorotic mottle virus |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9207886/ https://www.ncbi.nlm.nih.gov/pubmed/35757182 http://dx.doi.org/10.1186/s42483-022-00128-y |
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