一种快速检测桉树焦枯病菌(Cylindrocladium scoparium)的LAMP体系的建立及应用

Development of a loop-mediated isothermal amplification assay for detection of Cylindrocladium scoparium on Eucalyptus

摘要:

【目的】建立桉树焦枯病快速有效的环介导等温扩增反应LAMP检测技术。【方法】以桉树焦枯病原菌Cylindrocladium scoparium为研究对象，利用Primer Explorer V4.0设计软件，针对C. scoparium的beta-tubulin gene特异保守区域设计了一套LAMP特异性引物组(内引物FIP/BIP，外引物F3/B3，环引物LooPF/LooPB)，优化并建立了桉树焦枯病原菌的LAMP快速检测体系。【结果】LAMP反应体系为(50 μL)：Bst DNA polymerase (8 U) 1 μL；甜菜碱溶液(5 mol/L) 3.0 μL；dNTPs (2.5 mmol/L) 3.5 μL；10×Thermopol Ⅱ 2.5 μL；MgCl2 (100 mmol/L) 2 μL；FIP/BIP (40 μmol/L)各1 μL；F3/B3 (10 μmol/L)各0.5 μL；LooPF/LooPB (10 μmol/L)各1 μL；DNA模板2 μL；用ddH2O补足体积至50 μL。反应程序为：65 °C水浴锅中反应45 min，90 °C灭活5 min结束反应。特异性检测结果显示，供试的12个样本菌株LAMP产物可以采用肉眼观察、紫外灯照射、添加荧光染料SYBR Green I以及电泳检测条带4种不同的形式予以区分。DNA灵敏度检测结果显示，建立的LAMP体系检测灵敏度可达到40 μg/L，完全符合田间检测的要求。野外田间时效检测结果显示，无论是利用紫外检测还是电泳检测均可成功地检测出各地区不同生理小种的桉树焦枯病原菌C. scoparium，且检测效果明显。【结论】该LAMP检测是一项能够作为野外基层田间检测的重要技术。

Abstract:

[Objective] To establish a rapid detection technology LAMP for Eucalyptus dieback. [Methods] The pathogen against on Eucalyptus was studied. Based on the species-specific conserved region of beta-tubulin gene on Cylindrocladium scoparium, a set of primers (inner primer: FIP/BIP, outer primer: F3/B3, Loop primers: LooPF/LooPB) were designed, a LAMP assay for rapid detection of C. scoparium was developed, evaluated and optimized. [Results] The LAMP reaction system (50 μL) consisted of Bst DNA polymerase (8 U) 1 μL, Betaine solution (5 mol/L) 3.0 μL, dNTPs (2.5 mmol/L) 3.5 μL, 10×Thermopol Ⅱ 2.5 μL, MgCl2 (100 mmol/L) 2 μL, FIP/BIP (40 μmol/L) 1 μL, F3/B3 (10 μmol/L) 0.5 μL, LooPF/LooPB (10 μmol/L) 1 μL, DNA template 2 μL, with ddH2O up to the volume of 50 μL. Response procedures acted as the mixture was in 65 °C water bath pot for about 45 min first, and then inactivated 5 min under the 90 °C to end of reaction. Specificity assays showed that the amplification results of 12 test isolates can be distinguished though visual inspection, ultraviolet light, added fluorescent dye SYBR Green I, and electrophoresis test strip, and the 4 pathogens were positive, and that of the other 8 pathogens were negative. The detection limit of LAMP was 40 pg pure genomic DNA per 50 μL reaction, which fits entirely in the field test. The wild field effectiveness showed that the different physiological small kinds different regions of C. scoparium can be detected with the method of LAMP, meanwhile the ultraviolet light and electrophoresis test strip were significantly. [Conclusion] Summing up the above, the LAMP rapid detection can be applied in filed effectively.