A Fast and Sensitive Enzyme-Mediated Duplex Exponential Amplification Method for Field Detection of <i>Bursaphelenchus xylophilus</i>
The pinewood nematode (PWN), <i>Bursaphelenchus xylophilus</i>, is a pathogenic organism that causes pine wilt disease (PWD). To date, several molecular diagnostic methods have been developed; however, rapid, convenient, and inexpensive field diagnostic tools for detecting PWN are still...
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2025-05-01
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| author | Kai Guo Xinxin Ma Yiwu Fang Weijun Duan Yao Wu Zhenxin Hu Weimin Ye Jianfeng Gu |
| author_facet | Kai Guo Xinxin Ma Yiwu Fang Weijun Duan Yao Wu Zhenxin Hu Weimin Ye Jianfeng Gu |
| author_sort | Kai Guo |
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| description | The pinewood nematode (PWN), <i>Bursaphelenchus xylophilus</i>, is a pathogenic organism that causes pine wilt disease (PWD). To date, several molecular diagnostic methods have been developed; however, rapid, convenient, and inexpensive field diagnostic tools for detecting PWN are still limited. In this study, an enzyme-mediated duplex exponential amplification (EmDEA) method for detecting PWN from extracted nematodes or pinewood sawdust was developed and tested. This method comprised an EmDEA molecular test kit, which consisted of freeze-dried enzyme pellets that can be stored at room temperature (approximately 20–25 °C) for one year, a dry block heater, and a portable isothermal fluorescence amplification instrument. The whole procedure was completed within 30 min. The EmDEA assay could detect a single PWN at all life stages from a mixture of other nematode species or from pinewood sawdust. The detection limit was 10 copies (plasmid weight 32.66 ag) or 1/500 of that of a single adult PWN per reaction. Therefore, the EmDEA assay has potential applications in PWN detection in the field, as well as quarantine inspection in international trade. Moreover, modification of primers and probes will allow the rapid identification of other nematode species. |
| format | Article |
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| language | English |
| publishDate | 2025-05-01 |
| publisher | MDPI AG |
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| series | Horticulturae |
| spelling | doaj-art-a96b7e5825f94ca388c53b4402e8e08a2025-08-20T02:20:57ZengMDPI AGHorticulturae2311-75242025-05-0111660210.3390/horticulturae11060602A Fast and Sensitive Enzyme-Mediated Duplex Exponential Amplification Method for Field Detection of <i>Bursaphelenchus xylophilus</i>Kai Guo0Xinxin Ma1Yiwu Fang2Weijun Duan3Yao Wu4Zhenxin Hu5Weimin Ye6Jianfeng Gu7School of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, ChinaNingbo Key Laboratory of Port Biological and Food Safety Testing, Ningbo Customs Technology Center (Ningbo Inspection and Quarantine Science Technology Academy), 8 Huikang, Ningbo 315100, ChinaNingbo Key Laboratory of Port Biological and Food Safety Testing, Ningbo Customs Technology Center (Ningbo Inspection and Quarantine Science Technology Academy), 8 Huikang, Ningbo 315100, ChinaNingbo Key Laboratory of Port Biological and Food Safety Testing, Ningbo Customs Technology Center (Ningbo Inspection and Quarantine Science Technology Academy), 8 Huikang, Ningbo 315100, ChinaGeneVide Biotech Co., Ltd., Unit 201, Building A6, 218 Xinghu Street, Suzhou Industrial Park, Suzhou 215123, ChinaGeneVide Biotech Co., Ltd., Unit 201, Building A6, 218 Xinghu Street, Suzhou Industrial Park, Suzhou 215123, ChinaNematode Assay Section, Agronomic Division, North Carolina Department of Agriculture & Consumer Services, 4300 Reedy Creek Road, Raleigh, NC 27607, USANingbo Key Laboratory of Port Biological and Food Safety Testing, Ningbo Customs Technology Center (Ningbo Inspection and Quarantine Science Technology Academy), 8 Huikang, Ningbo 315100, ChinaThe pinewood nematode (PWN), <i>Bursaphelenchus xylophilus</i>, is a pathogenic organism that causes pine wilt disease (PWD). To date, several molecular diagnostic methods have been developed; however, rapid, convenient, and inexpensive field diagnostic tools for detecting PWN are still limited. In this study, an enzyme-mediated duplex exponential amplification (EmDEA) method for detecting PWN from extracted nematodes or pinewood sawdust was developed and tested. This method comprised an EmDEA molecular test kit, which consisted of freeze-dried enzyme pellets that can be stored at room temperature (approximately 20–25 °C) for one year, a dry block heater, and a portable isothermal fluorescence amplification instrument. The whole procedure was completed within 30 min. The EmDEA assay could detect a single PWN at all life stages from a mixture of other nematode species or from pinewood sawdust. The detection limit was 10 copies (plasmid weight 32.66 ag) or 1/500 of that of a single adult PWN per reaction. Therefore, the EmDEA assay has potential applications in PWN detection in the field, as well as quarantine inspection in international trade. Moreover, modification of primers and probes will allow the rapid identification of other nematode species.https://www.mdpi.com/2311-7524/11/6/602<i>Bursaphelenchus xylophilus</i>diagnosispine wilt diseaseisothermal fluorescence amplification |
| spellingShingle | Kai Guo Xinxin Ma Yiwu Fang Weijun Duan Yao Wu Zhenxin Hu Weimin Ye Jianfeng Gu A Fast and Sensitive Enzyme-Mediated Duplex Exponential Amplification Method for Field Detection of <i>Bursaphelenchus xylophilus</i> Horticulturae <i>Bursaphelenchus xylophilus</i> diagnosis pine wilt disease isothermal fluorescence amplification |
| title | A Fast and Sensitive Enzyme-Mediated Duplex Exponential Amplification Method for Field Detection of <i>Bursaphelenchus xylophilus</i> |
| title_full | A Fast and Sensitive Enzyme-Mediated Duplex Exponential Amplification Method for Field Detection of <i>Bursaphelenchus xylophilus</i> |
| title_fullStr | A Fast and Sensitive Enzyme-Mediated Duplex Exponential Amplification Method for Field Detection of <i>Bursaphelenchus xylophilus</i> |
| title_full_unstemmed | A Fast and Sensitive Enzyme-Mediated Duplex Exponential Amplification Method for Field Detection of <i>Bursaphelenchus xylophilus</i> |
| title_short | A Fast and Sensitive Enzyme-Mediated Duplex Exponential Amplification Method for Field Detection of <i>Bursaphelenchus xylophilus</i> |
| title_sort | fast and sensitive enzyme mediated duplex exponential amplification method for field detection of i bursaphelenchus xylophilus i |
| topic | <i>Bursaphelenchus xylophilus</i> diagnosis pine wilt disease isothermal fluorescence amplification |
| url | https://www.mdpi.com/2311-7524/11/6/602 |
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