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以硝酸纤维素膜为载体,对Dot-ELISA法的封闭条件、包被抗体浓度、点样量等反应条件进行优化,建立改良DAS-Dot-ELISA法快速检测西瓜细菌性果斑病菌。研究发现,以含乙二胺四乙酸二钠(EDTA)的脱脂奶粉液高温处理后用于封闭,可有效降低背景;轻微振荡可提高杂交效率,减少非特异性结合。改良DAS-Dot-ELISA可快速、经济的检测西瓜果斑病菌,灵敏度达1.9×105CFU/mL。在对两批次种子样品的检测中,改良DAS-Dot-ELISA法检测带菌率分别为8.0%和6.0%,与微孔板ELISA结果完全一致;对每粒种子的检测结果,改良DAS-Dot-ELISA法与微孔板ELISA吻合率平均达99.0%,显示较好的实用前景,同时为快速检测西瓜果斑病菌提供一种新途径。
The nitrocellulose membrane was used as a carrier to optimize the reaction conditions of Dot-ELISA, such as blocking conditions, coating antibody concentration and spotting volume, and to establish a rapid DAS-Dot-ELISA method for rapid detection of bacterial fruit spot pathogens in watermelon. The results showed that the skim milk solution containing ethylenediaminetetraacetic acid (EDTA) was used for blocking after high temperature treatment, which can effectively reduce the background; slight oscillation can improve the hybridization efficiency and reduce the non-specific binding. Improved DAS-Dot-ELISA can quickly and economically detect watermelon fruit fungus, the sensitivity of 1.9 × 105CFU / mL. The detection rate of modified DAS-Dot-ELISA method was 8.0% and 6.0% in the two batches of seed samples respectively, which was in good agreement with the results of microplate ELISA. For each seed detection result, DAS-Dot ELISA method and microplate ELISA anastomosis of an average of 99.0%, showing a good practical prospects, and for the rapid detection of watermelon fruit spot pathogen provide a new way.