薏苡黑穗病快速检测方法研究

Study on Rapid Detection Method of Coix lacryma-jobi Smut

以薏苡黑穗病菌粉胞内蛋白为免疫原,制备抗体,建立薏苡黑穗病ELISA检测方法。结果表明,测定纯化后抗体的最高效价为1∶800000,具特异性强;方阵试验测定抗原的最佳包被浓度为103 CFU/mL,抗体的工作浓度为1∶4000;优化ELISA检测条件,确定抗体4℃过夜(8~12 h)包被效果最好,选择1%酪蛋白作为抗体的封闭液,抗体的最佳封闭时间为1.5 h,抗体的最佳孵育时间为2.5 h,最佳底物作用时间为15 min;建立ELISA标准工作曲线,表明抗体的灵敏度为104 CFU/mL。ELISA检测田间采集的黑穗病病叶显示,抗体可与病叶提取液发生特异性反应,而与健康叶提取液呈阴性反应,由此可确定黑穗病的存在与否。该方法可用于田间薏苡黑穗病快速检测,以及时指导病害防治。

The antibody was prepared by the immunogen with the intracellular protein in Coix lacryma-jobi smut fungus powder,and the ELISA detection method was established.The results showed that the titer was 1∶800000 of the purified antibody,which had high specific.The square matrix test to determine the optimal coating concentration of the antigen was 103 CFU/mL,and the working concentration of the antibody was 1∶4000;the optimization ELISA detection conditions were antigen blocked for night(8-12 h)at 4℃,1%cottomin of the assay buffer blocked for 1.5 h,antigen incubated for 2.5 h,the reaction time of coloration was 15 min.The standard curves of ELISA was established,which showed that the antibody sensitivity was 104 CFU/mL.ELISA detection of smut diseased leaves collected in the field showed that the antibody could be specific reaction with the extracted solution of disease leaves,and it be negative reaction with extracted solution of healthy leaves,which could confirm the presence or absence of smut.The method can be applied to detect cucumber bacterial white spot disease in field and direct the disease control in time.