甘蔗高粱花叶病毒间接ELISA检测方法的建立和应用  

Establishment and Application of Indirect-ELISA for Detection of Sorghum mosaic virus in Sugarcane

作　　者：王凤林 廖永凤 李书巧 林垠孚 黄增飞 袁军涛[1] 陈保善[1,2] 温荣辉 

机构地区：[1]广西大学生命科学与技术学院,南宁530005 [2]亚热带农业生物资源保护与利用国家重点实验室,南宁530005

出　　处：《基因组学与应用生物学》2017年第10期4206-4211,共6页Genomics and Applied Biology

基　　金：广西回国基金重点项目(2013GXNSFCB019003);"教育发展资金(创优)区优质本科专业-生物技术"专项经费;广西蔗糖产业协同创新中心项目共同资助

摘　　要：利用人工合成的高粱花叶病毒P3蛋白的多肽片段作为抗原,制备多克隆抗体,利用免疫印迹对抗体的特异性进行鉴定,表明抗体具有高度特异性。通过方阵滴定法确定抗原和抗体的最佳工作浓度,并分别对包被时间、二抗工作浓度、孵育条件、底物显色时间等进行优化,从而建立了高粱花叶病毒(Sr MV)的间接ELISA高通量检测方法。通过测试确定了阴阳性结果判定的临界值,评估了该方法的灵敏度以及与RT-PCR检测法的符合度。所建立的间接ELISA检测方法的抗原、一抗、二抗最佳稀释度分别为1:4、1:600和1:5 000;最佳抗原包被条件为4℃、12 h;抗原抗体最佳孵育条件为37℃、60 min;二抗最佳孵育条件为37℃、45 min;最佳显色条件为37℃、12 min;此检测方法检测速度快、灵敏度高、特异性强,与RT-PCR法的符合度为87.00%。Polyclonal antibodies were prepared by using the polypeptide fragments of the synthetic Sorghum mosaic Virus P3 protein as antigen, and the specificity of the antibodies was identified by Western blotting, indicating that the antibodies were highly specific. Polyclonal antibody against the P3 protein of Sorghum mosaic virus（SrMV） was prepared by immunizing method of artificial peptide antigen. Western blotting analysis suggested that the polyclonal antibody can specifically recognized the P3 protein of Sr MV. The optimal working dilutions of antigen and antibody were determined by array titration, and then the indirect-ELISA method was established by further optimizing the incubate time, conditions of antibody, working dilution of second antibody, and visualization time of substrate. Finally, the critical value of positive and negative results were determined, the sensitivity of method and the conformity with the RT-PCR detection method were evaluated. The optimized dilutions of antigen, antibody, and second antibody of the established indirect-ELISA method were 1： 4, 1： 600, and 1： 5 000 respectively,and the optimized coating conditions of antigen were 4℃ for 12 h; The optimized incubating conditions of antigen and antibody were 37℃ for 60 min; The optimized incubating conditions of second antibody were 37℃ for 45 min;The optimized visualization conditions were 37℃ for 12 min. Results indicated that the indirect-ELISA method was rapid, sensitive, and specific, and the result conformity degree with the RT-PCR method was 87%.

关 键 词：高粱花叶病毒 间接ELISA 检测 

分 类 号：S435.661[农业科学—农业昆虫与害虫防治]