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| 改良DAS-Dot-ELISA检测西瓜细菌性果斑病菌 | |
| 引用本文： | 熊亮斌,刘箐,王天昌,高丽萍,王婧,刘姝彤,宋蕤,施颖波,王军平,文朝慧.改良DAS-Dot-ELISA检测西瓜细菌性果斑病菌[J].微生物学通报,2010,37(10):1551-1556. |
| 作者姓名： | 熊亮斌 刘箐 王天昌 高丽萍 王婧 刘姝彤 宋蕤 施颖波 王军平 文朝慧 |
| 作者单位： | 1. 兰州大学基础医学院,甘肃兰州,730000;甘肃出入境检验检疫局国家级外繁种子检疫重点实验室,甘肃兰州,730020 2. 甘肃出入境检验检疫局国家级外繁种子检疫重点实验室,甘肃兰州,730020;兰州慧盟生物科技有限公司,甘肃兰州,730010 3. 兰州慧盟生物科技有限公司,甘肃兰州,730010 4. 兰州大学基础医学院,甘肃兰州,730000 5. 甘肃农业大学食品科学学院,甘肃兰州,730070 6. 甘肃出入境检验检疫局国家级外繁种子检疫重点实验室,甘肃兰州,730020 |
| 基金项目： | 国家质量监督检验检疫总局项目(No. 2007IK259, 2009IK276); 甘肃省科技厅中小企业创新基金计划(No. 0802NCCA028) |
| 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1.9×105CFU/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。 |
| 关 键 词： | 西瓜细菌性果斑病菌 双抗体夹心Dot-ELISA 硝酸纤维素膜 快速检测 |
An Improved DAS Dot ELISA Method for Detection of Acidovorax avenae subsp. citrulli |
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| XIONG Liang-Bin,LIU Qing,WANG Tian-Chang,GAO Li-Ping,WANG Jing,LIU Shu-Tong,SONG Rui,SHI Ying-Bo,WANG Jun-Ping and WEN Zhao-Hui.An Improved DAS Dot ELISA Method for Detection of Acidovorax avenae subsp. citrulli[J].Microbiology,2010,37(10):1551-1556. | |
| Authors: | XIONG Liang-Bin LIU Qing WANG Tian-Chang GAO Li-Ping WANG Jing LIU Shu-Tong SONG Rui SHI Ying-Bo WANG Jun-Ping and WEN Zhao-Hui |
| Institution: | 1. School of Basic Medical Science, Lanzhou University, Lanzhou, Gansu 730000, China; 2. Gansu Entry-Exit Inspection and Quarantine Bureau, Lanzhou, Gansu 730020, China;2. Gansu Entry-Exit Inspection and Quarantine Bureau, Lanzhou, Gansu 730020, China; 3. Lanzhou Prajna-Union Biology Technique Ltd., Lanzhou, Gansu 730010, China;3. Lanzhou Prajna-Union Biology Technique Ltd., Lanzhou, Gansu 730010, China;1. School of Basic Medical Science, Lanzhou University, Lanzhou, Gansu 730000, China;4. College of Food Science and Engineering, Gansu Agricultural University, Lanzhou, Gansu 730070, China;2. Gansu Entry-Exit Inspection and Quarantine Bureau, Lanzhou, Gansu 730020, China;2. Gansu Entry-Exit Inspection and Quarantine Bureau, Lanzhou, Gansu 730020, China;2. Gansu Entry-Exit Inspection and Quarantine Bureau, Lanzhou, Gansu 730020, China;2. Gansu Entry-Exit Inspection and Quarantine Bureau, Lanzhou, Gansu 730020, China;2. Gansu Entry-Exit Inspection and Quarantine Bureau, Lanzhou, Gansu 730020, China |
| 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. |
| Keywords: | Acidovorax avenae subsp citrulli DAS-Dot-ELISA Nitrocellulose membrane Rapid detection |
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