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禾谷镰孢(Fusarium graminearum)和拟轮枝镰孢(Fusarium verticillioides)是引起玉米穗腐病的两种主要病原菌。为了建立这两种病原菌的快速和定量检测体系,本研究分别设计了针对F.graminearum和F.verticillioides的基因特异性引物,建立了双重PCR反应体系,并从DNA浓度、引物浓度和退火温度3个方面对反应体系进行了优化。结果表明,退火温度为54℃和58℃时扩增效果较好,DNA模版的检测灵敏度可以达到6.25 ng·μL~(-1);F.graminearum和F.verticillioides引物最佳终浓度配比为0.2μmol·L~(-1)/0.4μmol·L~(-1)。本研究建立的双重PCR对同时鉴别引起玉米穗腐病的两种主要镰孢菌提供了准确、快速、灵敏的检验检疫技术。
Fusarium graminearum and Fusarium verticillioides are the two major pathogens responsible for corn ear rot. In order to establish a rapid and quantitative detection system for these two pathogens, we designed two gene-specific primers for F.graminearum and F.verticillioides, respectively, and established a dual-PCR reaction system. From DNA concentration, primer concentration and annealing temperature 3 The aspects of the reaction system has been optimized. The results showed that the amplification efficiency was good at annealing temperature of 54 ℃ and 58 ℃, and the detection sensitivity of DNA template could reach 6.25 ng · μL -1. The optimum final concentration of F.graminearum and F.verticillioides primers was 0.2μmol·L -1 /0.4μmol·L -1. The dual PCR established in this study provided accurate, rapid and sensitive inspection and quarantine techniques for the simultaneous identification of two major Fusarium pathogens causing corn ear rot.