| 摘要: |
| 为快速鉴定Fusarium oxysporum f. sp. fragariae(Fof)引起的草莓枯萎病，主要采用环介导等温扩增技术(Loop-mediated isothermal amplification，LAMP)建立了Fof的快速检测体系，使用组织分离法和PCR法对LAMP技术辅佐验证。结果表明:25 μL体系中添加Buffer 2.50 μL、Mg2+(100 mmol/L)0.25 μL、dNTPs(10 mmol/L)1.00 μL、FIP和BIP(20 μmol/L)各1.00 μL、F3和B3(10 μmol/L)各0.50 μL、LoopF(10 μmol/L)0.75 μL、Bst DNA 聚合酶(8 U/μL)0.50 μL、Betaine(5 mol/L)1.00 μL、DNA模板1.00 μL和ddH2O 15.00 μL，反应结束后添加0.40 μL的SYBR Green I染料为最佳反应体系；该体系最适反应温度为62.9 ℃，最佳反应时间为60 min；特异性试验表明该体系能从16种草莓常见病原菌中特异性检测出Fof，最低检出限为45 pg/μL，比常规PCR检测效率高；随机抽取田间病害样品16株，采用LAMP体系检测罹病草莓根茎中的病原菌，其中12株感病草莓的病原菌为Fof，使用组织分离法和PCR法验证了LAMP检测结果的准确性；传统病原菌鉴定耗时约5~7 d，而LAMP检测1 d内便可完成。综上，本研究建立的LAMP检测体系为Fof准确快速检测及实际应用奠定了基础。 |
| 关键词: 草莓 草莓枯萎病 F.oxysporum f.sp.fragariae 环介导等温扩增技术 聚合酶链式反应 |
| DOI：10.11841/j.issn.1007-4333.2022.03.18 |
| 投稿时间：2021-04-21 |
| 基金项目:国家重点研发计划项目(2019YFD1001800) |
|
| Detection of the causal agent of strawberry Fusarium wilt by loop-mediated isothermal amplification |
|
HOU Shengfan,LIU Junjie,LI Xiaofeng,WANG Hongqing
|
| (College of Horticulture, China Agricultural University, Beijing 100193, China) |
| Abstract: |
| To rapidly identify strawberry Fusarium wilt caused by Fusarium oxysporum f. sp. fagariae (Fof), a rapid detection system of Fof was established based on loop-mediated isothermal amplification(LAMP). Tissue separation method and PCR analysis were used for LAMP verification. The results showed that: Buffer 2. 50 μL, Mg2+(100 mmol/L)0. 25 μL, dNTPs(10 mmol/L)1. 00 μL, FIP(20 μmol/L)1. 00 μL, BIP(20 μmol/L)1. 00 μL, F3(10 μmol/L)0. 50 μL, B3(10 μmol/L)0. 50 μL, LoopF(10 μmol/L)0. 75 μL, Bst DNA polymerase(8 U/μL)0. 50 μL, betaine(5 mol/L)1. 00 μL, DNA template 1. 00 μL ddH2O, 15. 00 μL and 0. 40 μL SYBR Green I dye added after the reaction was the most optimal reaction system for color rendering. The optimal reaction temperature was 62. 9 ℃ and the reaction time was 60 min. Specificity tests showed that the system could detect Fof from sixteen strawberry pathogens and the minimum detection limit was 45 pg/μL, which was basically the same as the detection limit by conventional PCR. Field disease samples were randomly selected, and the pathogenic fungi in sixteen strawberry root-stems were detected by LAMP system. The pathogenic fungi of twelve susceptible strawberry plants contained Fof. The accuracy of LAMP was verified by tissue separation and PCR. The traditional pathogen identification takes about 5-7 days to complete, while the LAMP complete detection can be completed within one day. In conclusion, the LAMP detection system established in this study lays a foundation for accurate and rapid detection of Fof and its practical application. |
| Key words: strawberry strawberry Fusarium wilt Fusarium oxysporum f. sp. fragariae loop-mediated isothermal amplification polymerase chain reaction |
