利用可视化蛋白芯片快速检测黄瓜绿斑驳花叶病毒

摘要

黄瓜绿斑驳花叶病毒(CGMMV)是瓜类作物上重要的病原病,需建立快捷、准确的检测方法.以硝酸纤维素膜为固相载体,采用 ELISA双抗夹心法建立了对CGMMV的可视化蛋白芯片检测方法,并对捕获抗体稀释倍数、样品孵育时间、酶标抗体孵育时间、显色温度等进行优化.结果表明,最佳检测条件为：捕获抗体稀释倍数1∶5,样品20℃孵育1 h,酶标抗体20℃孵育1 h,20℃ 15 min显色.以50批葫芦科种子为样品,对可视化蛋白芯片检测与DAS-ELISA检测进行了比较,其检测结果吻合率达98.0%；经 PCR检验,检测结果一致性良好.说明该方法能对植物病毒做出快速、准确的检测.%Cucumber green mottle mosaic virus (CGMMV)is one of the most important viruses in cu-curbit crops.Development and application of accurate and easy methods of CGMMV detection is essential for the prevention and control of associated diseases.An antibody macroarray procedure was developed and its usefulness for the detection of CGMMV was demonstrated in this study.The macroarray assay was based on a ‘sandwich’ELISA format,using a chromogenic dye,and consists of viral antibodies dotted di-rectly on nitrocellulose membrane (NCM).Using the conventional immunoassay ELISA technique as a benchmark,the optimized procedure was used to detect CGMMV.The results indicated that good reaction signals were based on coating antibody dilution factor 1∶5,sample incubated 1 h at 20 ℃,enzyme-linked an-tibody incubated 1 h at 20 ℃,substrate chromogenic reacted 15 min at 20 ℃.This method was further vali-dated by testing 50 lots of melon seeds,the coincidence rate was 98.0% between the results of antibody macroarray and that of ELISA.The results indicate that the developed antibody macroarray is sensitive and easy to use for diagnosis.