| 李本金 1, 刘小丽 1, 刘裴清 1, 王荣波 1, 翁启勇 1, 2*, 陈庆河 1, 2*.豇豆疫霉LAMP检测方法的建立[J].植物保护,2018,44(4):119-124. |
| 豇豆疫霉LAMP检测方法的建立 |
| Development of a LAMP assay for the detection of Phytophthora vignae |
| 投稿时间：2017-10-12 修订日期：2017-12-22 |
| DOI：10.16688/j.zwbh.2017389 |
| 中文关键词: 豇豆疫霉 内转录间隔区(ITS) 环介导等温扩增技术(LAMP) 分子检测 |
| 英文关键词:Phytophthora vignae ITS loop-mediated isothermal amplification (LAMP) molecular detection |
| 基金项目:福建省属公益类科研院所基本科研专项（2015R1024-2、2016R1023-1）；生物农药与化学生物学教育部重点实验室开放基金（Keylab2016-01）；福建省农业科学院植物保护科技创新团队（STIT2017-1-8） |
| 作者 | 单位 | | 李本金 1, 刘小丽 1, 刘裴清 1, 王荣波 1, 翁启勇 1, 2*, 陈庆河 1, 2* | 1. 福建省农业科学院植物保护研究所, 福建省作物有害生物监测与治理重点实验室, 福州 350013
2. 福建农林大学, 闽台作物有害生物生态防控国家重点实验室, 福州 350002 |
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| 中文摘要: |
| 本研究以豇豆疫霉Phytophthora vignae Purss核糖体内转录间隔区(internal transcribed spacer, ITS)序列为靶标片段, 设计4条特异性引物, 建立了环介导等温扩增(LAMP)检测方法。特异性检测结果表明：8株不同地理来源的豇豆疫霉菌株LAMP检测均为阳性(绿色), 扩增产物用2.0%琼脂糖凝胶电泳出现特有的梯形条带, 而其他11种卵菌近缘种及12种常见病原真菌和细菌共42个菌株均未观察到这些现象。灵敏度分析显示：该方法检测灵敏度在DNA水平上可达到100 fg/25 μL。采用LAMP方法对福建建瓯和宁德采集的62份疑似豇豆疫病病株样本进行检测, 并用组织分离方法进行验证。结果表明, LAMP和组织分离方法的检出率分别为67.7%(42/62)和61.3%(38/62)。综合以上结果, LAMP方法具有特异性强、灵敏度高、快速高效、操作简单的特点, 适合基层部门用于田间豇豆疫霉快速检测。 |
| 英文摘要: |
| A set of four primers for loop-mediated isothermal amplification (LAMP) were designed according to the internal transcribed spacer (ITS) sequence of Phytophthora vignae. The LAMP reaction conditions were optimized. The specificity, the sensitivity were assayed and the diseased cowpea tissues were detected using the optimized LAMP method. The specificity analysis results showed that eight P.vignae isolates from different counties of Fujian were positive (green color in reaction tube) and the reaction products showed typical ladder-like banding pattern on 2.0% agarose gel, but 29 isolates of 11 closely related species of oomycetes and 13 isolates of other plant pathogenic fungi and bacteria did not. Sensitivity analysis indicated that 100 fg DNA in 25 μL PCR reaction solution could be detected using the developed LAMP detection system. A total of 62 cowpea suspected infected samples collected from Jian’ou and Ningde County were examined by the LAMP detection system and verified using the conventional tissue isolation method. The results showed that the detection rates were 67.7% (42 of 62) by the LAMP assay and 61.3% (38 of 62) for the conventional culture method, respectively. We concluded that the developed LAMP assay was specific, sensitive, fast and simple, and can be used in field detection of P.vignae. |
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