应用 PCR 法对小麦叶疫病菌的检测

摘要

Background] After the Alternaria leaf blight infection of wheat in China in the 1960s, which spread rapidly and caused serious harm to the local economy, it become a constant threat for the production of China′s wheat.[Method] We designed specific primers in order to establish a rapid PCR detection method for Alternaria triticina Prasada & Prabhu.The fungal universal primers ITS4/ITS5 were used to amplify the A.triticina by PCR.The PCR products were cloned and sequenced .The bacteria specific primers LJY1 and LJY2 were designed by DNAMAN software.And the reaction system was optimized.[Results] PCR reaction was set up:25 mmol· L-1 MgCl2 2.5μL, 10 mmol· L-1 dNTP 1.0μL, 10μmol· L-1 primers 0.5μL respectively, the template of DNA was 8 ng, the best annealing temperature was 57.6℃.The rapid PCR detection method for the species was established.[Conclusion and significance] The testing of the method for detection on the 9 different strains showed that the primer can accurately distinguish wheat leaf blight from other Alternaria fungi.The results for the rapid detection of wheat leaf blight provide an experimental basis, which can effectively used to prevent the bacteria from entering China′s import and export trade of wheat.%【背景】小麦叶疫病菌于20世纪60年代入侵我国后，迅速传播扩散并在局部区域造成严重危害，对我国小麦的健康发展构成了巨大威胁。【方法】设计出检测小麦叶疫病菌的特异性引物，建立快速检测该病菌的PCR方法。用真菌通用引物ITS4／ITS5对小麦叶疫病菌进行PCR扩增，将扩增产物进行克隆和测序，使用DNAMAN软件设计出检测该病菌的特异性引物 LJY1和LJY2，优化PCR反应体系。【结果】建立了该病菌的PCR检测方法，PCR反应体系：25 mmol· L－1 MgCl22．5μL，10 mmol· L－1 dNTP 1．0μL，10μmol· L－1引物各0．5μL，DNA模板8 ng，最佳退火温度57．6℃。【结论与意义】该方法可以准确地将小麦叶疫病菌与其他链格孢属的真菌区分开。本研究结果为小麦叶疫病的快速检测提供了依据，能够有效防止该病菌在小麦进出口贸易中传入我国。