改良DAS-Dot-ELISA检测西瓜细菌性果斑病菌

An Improved DAS Dot ELISA Method for Detection of Acidovorax avenae subsp. citrulli

摘要:

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 × 105 CFU/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。

Abstract:

Based on nitrocellulose membrane, we optimized the blocking condition, the volume and concentration of coating antibody. Then, an improved Double Antibody Sandwich Dot Enzyme Linked Immunosorbent Assay (DAS-Dot-ELISA) for Acidovorax avenae subsp. citrulli, the casual agent of bacterial fruit blotch of watermelon, was established. Use adding Disodium Ethylenediamine Tetraacetic Acid (EDTA) in nonfat dry milk treated with high temperature as blocking solution can effectively lower the background. Shaking gently can enhance hybridization and reduce the nonspecific binding. This improved DAS-Dot-ELISA assay detected Acidovorax avenae subsp. citrulli as low as 1.9 × 105 CFU/mL quickly and economically. On the detection of two batches seed sample, the germ-carrying rate with improved DAS-Dot-ELISA was 8.0%, 6.0% which was completely coincident with microwell plate ELISA. On the result of each seed, this improved method was coincided with microwell plate ELISA in 99%, showed a satisfactory prospect and provided a new rapid assay for the detection of Acidovorax avenae subsp. citrulli.