| 段维军1, 2*, 李雪莲1, 2, 吕燕1, 3, 张晓梅4.长孢轮枝菌Taq〖KG-*4〗Man-MGB探针实时荧光PCR快速检测方法[J].植物保护,2023,49(4):224-232. |
| 长孢轮枝菌Taq〖KG-*4〗Man-MGB探针实时荧光PCR快速检测方法 |
| Development of TaqMan-MGB fluorescent real-time PCR assay for the detection of Verticillium longisporum |
| 投稿时间：2022-06-15 修订日期：2022-08-24 |
| DOI：10.16688/j.zwbh.2022332 |
| 中文关键词: 长孢轮枝菌 实时荧光PCR TaqMan-MGB探针 检测 |
| 英文关键词:Verticillium longisporum real-time fluorescent PCR TaqMan-MGB probe detection |
| 基金项目:国家重点研发计划(2022YFF0608804); 青海省基础研究计划(2022-ZJ-726); 宁波市科研项目(2022S010) |
| 作者 | 单位 | E-mail | | 段维军1, 2*, 李雪莲1, 2, 吕燕1, 3, 张晓梅4 | 1. 宁波检验检疫科学技术研究院, 宁波315012
2. 宁波海关, 宁波315012
3. 宁波中盛产品检测有限公司, 宁波315012
4. 西宁市蔬菜技术服务中心, 西宁810016 | weijunduan@tom.com |
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| 中文摘要: |
| 长孢轮枝菌是一种在我国局部地区新近出现且危害性极大的植物病原真菌?根据长孢轮枝菌及其近似种的actin序列差异, 设计并合成特异性引物和探针, 建立了长孢轮枝菌的实时荧光PCR检测方法?特异性试验结果表明, 该检测方法能特异性检测长孢轮枝菌; 灵敏度试验结果表明, 最低检测限量为10 μL反应体系中总DNA含量10 pg; 实时荧光PCR优化反应条件为引物终浓度0.8 μmol/L, 探针终浓度0.8 μmol/L, 优化后的整个反应过程约1 h?实际样品检测结果表明, 该方法可用于疑似受长孢轮枝菌侵染的萝卜样品检测与初筛?此方法快速?灵敏, 检测过程完全闭管, 无需PCR后续处理, 为早期快速检测长孢轮枝菌提供了重要参考? |
| 英文摘要: |
| Verticillium longisporum is a newly occurring and important plant pathogenic fungus in local areas of China. A species-specific real-time polymerase chain reaction assay was developed for the detection of V.longisporum. A pair of specific primers and a TaqMan-MGB probe were designed and synthesized according to the difference of actin sequence between V.longisporum and related species. A novel real-time fluorescent PCR was established to detect V.longisporum. The minimal detectable concentration of targeted DNA was 10 pg in 10 μL reaction mixture. Both optimal primer concentration and probe concentration were 0.8 μmol/L. The method could be used for the detection and preliminary screening of the samples suspected of carrying V.longisporum. The optimized detection method could be completed within 1 h. The method was rapid, sensitive and completed within a single tube, without post-PCR handling of the amplification products. The method provided a valuable tool for early rapid detection of V. longisporum. |
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