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【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Loo PF/Loo PB),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(50μL):Bst DNA polymerase(8 U)1μL;甜菜碱溶液(5 mol/L)3.0μL;d NTPs(2.5 mmol/L)3.5μL;10×ThermopolⅡ2.5μL;Mg Cl2(100 mmol/L)2μL;FIP/BIP(40μmol/L)各1μL;F3/B3(10μmol/L)各0.5μL;Loo PF/Loo PB(10μmol/L)各1μL;DNA模板2μL;用dd H2O补足体积至50μL。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40μg/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。
【Objective】 To establish a rapid and efficient loop-mediated isothermal amplification reaction LAMP detection technique for eucalyptus scorch. 【Method】 Cylindrocladium scoparium was used as a research object. A set of LAMP-specific primer sets (inner primers FIP / BIP, Primer Explorer V4.0) were designed according to the specific conserved region of Beta-tubulin gene in C. scoparium. F3 / B3 and Loo PF / Loo PB) were used to optimize the LAMP rapid detection system of eucalyptus. 【Result】 The reaction system of LAMP was (50 μL): 1 μL of Bst DNA polymerase (8 U); 3.0 μL of 5 mol / L betaine; 3.5 μL of dNTPs (2.5 mmol / L); 2.5 μL of 10 × ThermopolⅡ 1 μL each of FIP / BIP (40 μmol / L), 0.5 μL each of F3 / B3 (10 μmol / L), and 1 μL each of Loo PF / Loo PB dd H2O make up volume to 50 μL. Reaction program: 65 ° C water bath for 45 min, 90 ° C inactivated 5 min the reaction. Specificity test results showed that the LAMP product of the 12 sample strains tested can be distinguished by naked eye observation, UV lamp irradiation, addition of fluorescent dye SYBR Green I and electrophoresis detection bands in four different forms. The results of DNA sensitivity test showed that the detection sensitivity of LAMP system was up to 40μg / L, which fully met the requirements of field testing. Field field aging test results show that C.scoparium can be successfully detected in different physiological races of Eucalyptus species either in UV or electrophoresis. 【Conclusion】 This LAMP assay is an important technique that can be used as field detection in field.