CRISPR/Cas12a-based method coupled with isothermal amplification to identify Alternaria spp. isolated from wheat grain samples
Alternaria fungal species are considered major plant pathogens, infecting various crops and resulting in significant agricultural losses. Additionally, these species can contaminate grain with multiple mycotoxins that are harmful to humans and animals. Efficient pest management relies on timely dete...
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| Language: | English |
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Frontiers Media S.A.
2025-01-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1468336/full |
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| author | Aisha Shaizadinova Aisha Shaizadinova Meruyert Amanzholova Meruyert Amanzholova Irina Rukavitsina Sailau Abeldenov Anuar Rysbekovich Zhumakayev |
| author_facet | Aisha Shaizadinova Aisha Shaizadinova Meruyert Amanzholova Meruyert Amanzholova Irina Rukavitsina Sailau Abeldenov Anuar Rysbekovich Zhumakayev |
| author_sort | Aisha Shaizadinova |
| collection | DOAJ |
| description | Alternaria fungal species are considered major plant pathogens, infecting various crops and resulting in significant agricultural losses. Additionally, these species can contaminate grain with multiple mycotoxins that are harmful to humans and animals. Efficient pest management relies on timely detection and identification of phytopathogens in plant and grain samples, facilitating prompt selection of a crop protection strategy. Conventional identification tools, such as morphological characterization and identification based on polymerase chain reaction (PCR)-based methods, are time-consuming and laboratory-bound, limiting their implementation for on-site diagnostics essential in the agricultural industry. Isothermal amplification methods, including nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA), enable nucleic acid amplification at constant temperatures, making them ideal for point-of-care diagnostics without the need for thermal cycling equipment. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a)-based identification, coupled with such isothermal amplification methods, represents an emerging nucleic acid-based technology for detecting plant pathogens at high accuracy and sensitivity. This study aimed to develop a CRISPR/Cas12a-based method integrated with RPA amplification for specific detection of Alternaria spp. isolated from wheat grain samples. The developed method targeted the β-tubulin gene was successfully identified Alternaria strains within a 20-min RPA amplification followed by a 30-min CRISPR/Cas12a reaction and visualization of results. Specificity test included pathogenic fungal species commonly hosted wheat grain, such as Fusarium spp. Bipolaris sorokiniana, and Nigrospora oryzae revealed high specificity of the method for Alternaria species. Furthermore, the method exhibited high sensitivity, detecting Alternaria DNA down to 100 copies, validated by real-time fluorescence readout. A fluorescence assay was employed to visualize the results of RPA and CRISPR/Cas12a reaction, demonstrating substantial implementation potential of the method in point-of-care detection of Alternaria spp. In conclusion, we present the CRISPR/Cas12a-based method as a potentially sustainable approach for the rapid, precise, and specific nucleic-acid-based identification of Alternaria species in grain samples. |
| format | Article |
| id | doaj-art-2b50c97bae914729b25e35d6ac0b576c |
| institution | Kabale University |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj-art-2b50c97bae914729b25e35d6ac0b576c2025-01-15T06:10:31ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-01-011510.3389/fmicb.2024.14683361468336CRISPR/Cas12a-based method coupled with isothermal amplification to identify Alternaria spp. isolated from wheat grain samplesAisha Shaizadinova0Aisha Shaizadinova1Meruyert Amanzholova2Meruyert Amanzholova3Irina Rukavitsina4Sailau Abeldenov5Anuar Rysbekovich Zhumakayev6Laboratory of Molecular Biotechnology, National Center for Biotechnology, Astana, KazakhstanFaculty of Biology and Biotechnology, Al-Farabi Kazakh National University, Almaty, KazakhstanLaboratory of Molecular Biotechnology, National Center for Biotechnology, Astana, KazakhstanFaculty of Biology and Biotechnology, Al-Farabi Kazakh National University, Almaty, KazakhstanLaboratory of Microbiology, A.I. Barayev Research and Production Centre for Grain Farming, Shortandy-1, KazakhstanLaboratory of Molecular Biotechnology, National Center for Biotechnology, Astana, KazakhstanLaboratory of Molecular Biotechnology, National Center for Biotechnology, Astana, KazakhstanAlternaria fungal species are considered major plant pathogens, infecting various crops and resulting in significant agricultural losses. Additionally, these species can contaminate grain with multiple mycotoxins that are harmful to humans and animals. Efficient pest management relies on timely detection and identification of phytopathogens in plant and grain samples, facilitating prompt selection of a crop protection strategy. Conventional identification tools, such as morphological characterization and identification based on polymerase chain reaction (PCR)-based methods, are time-consuming and laboratory-bound, limiting their implementation for on-site diagnostics essential in the agricultural industry. Isothermal amplification methods, including nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA), enable nucleic acid amplification at constant temperatures, making them ideal for point-of-care diagnostics without the need for thermal cycling equipment. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a)-based identification, coupled with such isothermal amplification methods, represents an emerging nucleic acid-based technology for detecting plant pathogens at high accuracy and sensitivity. This study aimed to develop a CRISPR/Cas12a-based method integrated with RPA amplification for specific detection of Alternaria spp. isolated from wheat grain samples. The developed method targeted the β-tubulin gene was successfully identified Alternaria strains within a 20-min RPA amplification followed by a 30-min CRISPR/Cas12a reaction and visualization of results. Specificity test included pathogenic fungal species commonly hosted wheat grain, such as Fusarium spp. Bipolaris sorokiniana, and Nigrospora oryzae revealed high specificity of the method for Alternaria species. Furthermore, the method exhibited high sensitivity, detecting Alternaria DNA down to 100 copies, validated by real-time fluorescence readout. A fluorescence assay was employed to visualize the results of RPA and CRISPR/Cas12a reaction, demonstrating substantial implementation potential of the method in point-of-care detection of Alternaria spp. In conclusion, we present the CRISPR/Cas12a-based method as a potentially sustainable approach for the rapid, precise, and specific nucleic-acid-based identification of Alternaria species in grain samples.https://www.frontiersin.org/articles/10.3389/fmicb.2024.1468336/fullAlternaria detectionwheat diseasesCRISPR diagnosticsCas12afungal plant pathogens |
| spellingShingle | Aisha Shaizadinova Aisha Shaizadinova Meruyert Amanzholova Meruyert Amanzholova Irina Rukavitsina Sailau Abeldenov Anuar Rysbekovich Zhumakayev CRISPR/Cas12a-based method coupled with isothermal amplification to identify Alternaria spp. isolated from wheat grain samples Frontiers in Microbiology Alternaria detection wheat diseases CRISPR diagnostics Cas12a fungal plant pathogens |
| title | CRISPR/Cas12a-based method coupled with isothermal amplification to identify Alternaria spp. isolated from wheat grain samples |
| title_full | CRISPR/Cas12a-based method coupled with isothermal amplification to identify Alternaria spp. isolated from wheat grain samples |
| title_fullStr | CRISPR/Cas12a-based method coupled with isothermal amplification to identify Alternaria spp. isolated from wheat grain samples |
| title_full_unstemmed | CRISPR/Cas12a-based method coupled with isothermal amplification to identify Alternaria spp. isolated from wheat grain samples |
| title_short | CRISPR/Cas12a-based method coupled with isothermal amplification to identify Alternaria spp. isolated from wheat grain samples |
| title_sort | crispr cas12a based method coupled with isothermal amplification to identify alternaria spp isolated from wheat grain samples |
| topic | Alternaria detection wheat diseases CRISPR diagnostics Cas12a fungal plant pathogens |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1468336/full |
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