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| H5N1禽流感病毒环介导等温扩增快速检测方法的建立 | |
| 引用本文： | 张坤,黄伟,李刚. H5N1禽流感病毒环介导等温扩增快速检测方法的建立[J]. 生物技术通讯, 2009, 20(2): 217-220. DOI: 10.3969/j.issn.1009-0002.2009.02.020 |
| 作者姓名： | 张坤 黄伟 李刚 |
| 作者单位： | 1. 西南大学,动物科技学院,重庆,400715;中国农业科学院,北京畜牧兽医研究所,北京,100093 2. 西南大学,动物科技学院,重庆,400715 3. 中国农业科学院,北京畜牧兽医研究所,北京,100093;动物营养学国家重点实验室,北京,100093 |
| 基金项目： | 联合国FAO/IAEA项目，中央级公益性科研院所基本科研业务费专项基金 |
| 摘 要： | 目的：建立H5N1禽流感病毒环介导等温扩增（LAMP）快速检测方法。方法：从GenBank中获得H5N1禽流感病毒血凝素（HA）基因序列,应用DNAStar软件MegAlign程序分析其序列,利用PrimerExplorerV3软件在序列保守区域设计LAMP引物,即外引物、内引物和环引物,同时以所克隆的阳性质粒为模板,对试验中的几个重要参数进行优化。结果：LAMP检测方法对H5N1禽流感病毒的灵敏度达到4～6个拷贝,其引物对于H1、H9亚型禽流感病毒和新城疫病毒无非特异性扩增,表现出良好的H5亚型特异性。结论：建立的H5N1禽流感病毒环介导等温扩增快速检测方法灵敏度高、特异性强、重复性好,为快速检测禽流感病毒提供了新方法和新思路。 |
| 关 键 词： | H5N1禽流感病毒 环介导等温扩增 条件优化 |
Development of Rapid Assay for Avian Influenza A(H5N1) Virus by Loop-Mediated Isothermal Amplification Method |
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| ZHANG Kun,HUANG Wei,LI Gang. Development of Rapid Assay for Avian Influenza A(H5N1) Virus by Loop-Mediated Isothermal Amplification Method[J]. Letters in Biotechnology, 2009, 20(2): 217-220. DOI: 10.3969/j.issn.1009-0002.2009.02.020 | |
| Authors: | ZHANG Kun HUANG Wei LI Gang |
| Affiliation: | 1. Animal Medicine, Animal Science and Technology College, Southwest University, Chongqing 400715; 2. Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, Beijing 100093; 3. State Key Laboratory of Animal Nutrition, Beijing 100093; China ) |
| Abstract: | Objective： To set up loop-mediated isothermal amplification（LAMP） method for quick detection of H5N1 avian influenza A virus. Methods： The hemagglutinin gene of avian influenza virus subtype H5 was downloaded from Gen- Bank and analyzed using MegAlign tools of software DNAStar. The outer, inner and loop primers of LAMP were then designed according to their conservative region using software PrimerExplorer V3. The fragment of subtype H5 sequence was also amplified and cloned to serve as the positive standard and optimized its LAMP reaction condition and reaction system in this experiment. Results： The results showed that the LAMP assay had a detection limit of 4-6 DNA copies. The primers used in this assay showed the specificity for H5N1 avian influenza A virus and did not amplify H1 virus or H9 or New Castle disease virus. Conclusion： It is proved that LAMP method is a reliable and accurate assay for quick detection of H5N1 virus. This assay offers a new method and idea for detecting avian influenza virus. |
| Keywords: | H5N1 avian influenza A virus loop-mediated isothermal amplification condition optimization |
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