基于环介导等温扩增技术的草莓枯萎病检测

Detection of the causal agent of strawberry Fusarium wilt by loop-mediated isothermal amplification

为快速鉴定Fusariumoxysporum 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-mediatedisothermalamplification,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:25μL体系中添加Buffer2.50μL、Mg^(2+)(100 mmol/L)0.25μL、dNTPs(10mmol/L)1.00μL、FIP和BIP(20μmol/L)各1.00μL、F3和B3(10μmol/L)各0.50μL、LoopF(10μmol/L)0.75μL、BstDNA聚合酶(8U/μL)0.50μL、Betaine(5mol/L)1.00μL、DNA模板1.00μL和ddH_(2)O15.00μL,反应结束后添加0.40μL的SYBR GreenⅠ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60min;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45pg/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5~7d,而LAMP检测1d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。

To rapidly identify strawberry Fusarium wilt caused by Fusarium oxysporumf.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 2.50μL,Mg^(2+)(100 mmol/L)0.25μL,dNTPs(10 mmol/L)1.00μL,FIP(20μmol/L)1.00μL,BIP(20μmol/L)1.00μL,F3(10μmol/L)0.50μL,B3(10μmol/L)0.50μL,LoopF(10μmol/L)0.75μL,Bst DNA polymerase(8 U/μL)0.50μL,betaine(5 mol/L)1.00μL,DNA template 1.00μL ddH_(2)O,15.00μL and 0.40μL SYBR GreenⅠdye 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.