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利用棉花黄萎病菌ITS区特异性引物建立了棉花黄萎病菌的实时荧光定量PCR检测方法。利用实时荧光PCR体系,以324 bp的PCR扩增产物构建了标准曲线并对该方法的灵敏度、特异性和重复性进行了评估。结果表明:该方法特异性良好,检测灵敏度为100 copies·μL-1,标准曲线的相关系数为0.994,扩增效率为91.5%。利用建立的检测方法对转基因棉田及常规棉田土壤样本进行检测,结果表明转基因棉田中棉花黄萎病菌数量显著高于常规棉田,与实际观测到的现象一致,也证明了本方法的可行性。因此,本研究建立的棉花黄萎病菌检测方法具有灵敏度高、重复性好等特点,为棉花的种植及病害防治提供了有效的检测手段。
The detection of Verticillium dahliae in cotton by Verticillium dahliae ITS-specific primers was established by real-time fluorescence quantitative PCR. Using real-time fluorescence PCR system, a standard curve was constructed with 324 bp PCR amplification products and the sensitivity, specificity and repeatability of the method were evaluated. The results showed that the specificity of the method was good, the detection sensitivity was 100 copies · μL-1, the correlation coefficient of standard curve was 0.994, and the amplification efficiency was 91.5%. The results showed that the amount of Verticillium dahliae in cotton field was significantly higher than that in conventional cotton field. The results were consistent with the observed ones, which also proved the feasibility of this method. Therefore, the detection method of Verticillium dahliae in this study has the characteristics of high sensitivity, good repeatability and so on, which provides an effective method for the cotton plantation and disease prevention and control.