Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12
Pine wilt disease is caused by the pine wood nematode (Bursaphelenchus xylophilus) and leads to wilting and death of pines. It is one of the most damaging diseases of pines worldwide. Therefore, accurate and rapid detection methods are of great importance for the control of B. xylophilus. Traditiona...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-12-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2022.1075838/full |
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| author | Xiang Wang Lai-Fa Wang Ye-Fan Cao Yan-Zhi Yuan Jian Hu Zu-Hai Chen Fei Zhu Xi-Zhuo Wang |
| author_facet | Xiang Wang Lai-Fa Wang Ye-Fan Cao Yan-Zhi Yuan Jian Hu Zu-Hai Chen Fei Zhu Xi-Zhuo Wang |
| author_sort | Xiang Wang |
| collection | DOAJ |
| description | Pine wilt disease is caused by the pine wood nematode (Bursaphelenchus xylophilus) and leads to wilting and death of pines. It is one of the most damaging diseases of pines worldwide. Therefore, accurate and rapid detection methods are of great importance for the control of B. xylophilus. Traditional detection methods have some problems, such as being time-consuming and requiring expensive instruments. In this study, the loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) were used to establish a set of intelligent detection and analysis system for B. xylophilus, called LAMP-CRISPR/Cas12a analysis, which integrated field sampling, rapid detection and intelligent control analysis. The process can be completed within 1 hour, from sample pretreatment and detection to data analysis. Compared with the single LAMP method, the LAMP-CRISPR/Cas12a assay uses species-specific fluorescence cleavage to detect target amplicons. This process confirms the amplicon identity, thereby avoiding false-positive results from non-specific amplicons, and the large amounts of irrelevant background DNA do not interfere with the reaction. The LAMP-CRISPR/Cas12a assay was applied to 46 pine wood samples and the samples carrying B. xylophilus nematodes were successfully identified. To meet the needs of different environments, we designed three methods to interpret the data: 1) naked eye interpretation; 2) lateral flow biosensor assay; and 3) integrated molecular analysis system to standardize and intellectualize the detection process. Application of the B. xylophilus detection and analysis system will reduce the professional and technical requirements for the operating environment and operators and help to ensure the accuracy of the detection results, which is important in grass-root B. xylophilus detection institutions. |
| first_indexed | 2024-04-12T01:16:03Z |
| format | Article |
| id | doaj.art-f047dd365f274b04ad81e5385958cab9 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-04-12T01:16:03Z |
| publishDate | 2022-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-f047dd365f274b04ad81e5385958cab92022-12-22T03:53:58ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2022-12-011310.3389/fpls.2022.10758381075838Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12Xiang Wang0Lai-Fa Wang1Ye-Fan Cao2Yan-Zhi Yuan3Jian Hu4Zu-Hai Chen5Fei Zhu6Xi-Zhuo Wang7Key Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, ChinaKey Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, ChinaKey Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, ChinaKey Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, ChinaKey Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, ChinaJingning County Forest Resources Management Center, Lishui, ChinaHangzhou Linping District Forest Resources Protection and Management Station, Hangzhou, ChinaKey Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, ChinaPine wilt disease is caused by the pine wood nematode (Bursaphelenchus xylophilus) and leads to wilting and death of pines. It is one of the most damaging diseases of pines worldwide. Therefore, accurate and rapid detection methods are of great importance for the control of B. xylophilus. Traditional detection methods have some problems, such as being time-consuming and requiring expensive instruments. In this study, the loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) were used to establish a set of intelligent detection and analysis system for B. xylophilus, called LAMP-CRISPR/Cas12a analysis, which integrated field sampling, rapid detection and intelligent control analysis. The process can be completed within 1 hour, from sample pretreatment and detection to data analysis. Compared with the single LAMP method, the LAMP-CRISPR/Cas12a assay uses species-specific fluorescence cleavage to detect target amplicons. This process confirms the amplicon identity, thereby avoiding false-positive results from non-specific amplicons, and the large amounts of irrelevant background DNA do not interfere with the reaction. The LAMP-CRISPR/Cas12a assay was applied to 46 pine wood samples and the samples carrying B. xylophilus nematodes were successfully identified. To meet the needs of different environments, we designed three methods to interpret the data: 1) naked eye interpretation; 2) lateral flow biosensor assay; and 3) integrated molecular analysis system to standardize and intellectualize the detection process. Application of the B. xylophilus detection and analysis system will reduce the professional and technical requirements for the operating environment and operators and help to ensure the accuracy of the detection results, which is important in grass-root B. xylophilus detection institutions.https://www.frontiersin.org/articles/10.3389/fpls.2022.1075838/fullBursaphelenchus xylophilusnucleic acid detectionCRISPR-Cas12aLAMPrapid diagnostic |
| spellingShingle | Xiang Wang Lai-Fa Wang Ye-Fan Cao Yan-Zhi Yuan Jian Hu Zu-Hai Chen Fei Zhu Xi-Zhuo Wang Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12 Frontiers in Plant Science Bursaphelenchus xylophilus nucleic acid detection CRISPR-Cas12a LAMP rapid diagnostic |
| title | Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12 |
| title_full | Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12 |
| title_fullStr | Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12 |
| title_full_unstemmed | Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12 |
| title_short | Bursaphelenchus xylophilus detection and analysis system based on CRISPR – Cas12 |
| title_sort | bursaphelenchus xylophilus detection and analysis system based on crispr cas12 |
| topic | Bursaphelenchus xylophilus nucleic acid detection CRISPR-Cas12a LAMP rapid diagnostic |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2022.1075838/full |
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