| 陈 杰, 朱天辉*.核桃枝枯病小新壳梭孢巢式PCR检测方法的建立[J].植物保护,2018,44(3):124-129. |
| 核桃枝枯病小新壳梭孢巢式PCR检测方法的建立 |
| Establishment of nested PCR method for detecting the branch dieback of walnut caused by Neofusicoccum parvum |
| 投稿时间：2017-07-11 修订日期：2017-09-12 |
| DOI：10.16688/j.zwbh.2017262 |
| 中文关键词: 核桃枝枯病 小新壳梭孢菌 分子检测 巢式PCR |
| 英文关键词:walnut blight Neofusicoccum parvum molecular detection technology nested PCR |
| 基金项目:中国博士后科学基金(2016M602705) |
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| 中文摘要: |
| 近年来, 小新壳梭孢 Neofusicoccum parvum 导致的核桃枝枯病屡有报道, 该病防治困难, 发病率高, 是核桃的一种灾难性病害。为建立 N. parvum 的快速、灵敏的分子检测体系, 根据 N. parvum 的几丁质合酶1基因(CHS1)及其前后100 bp序列, 分别设计了两对特异性引物CT-WK3-S/CT-WK3-A和CT-N-S/CT-N-A, 建立了 N. parvum 的巢式PCR检测方法。结果表明, 建立的体系可以从不同来源的5个 N. parvum 菌株中特异性地扩增出97 bp的目的条带, 灵敏度达30 fg/μL, 是常规PCR的10倍, 而从其近缘种葡萄座腔菌属 Botryosphaeria sp. 的病原菌及其他供试菌株均未扩增出任何条带。以感染 N. parvum 的核桃组织总DNA为模板进行检测, 可以在发病早期检测出 N. parvum 的存在。本研究建立的巢式PCR体系可以为核桃枝枯病的田间检测提供思路。 |
| 英文摘要: |
| The branch dieback of walnut caused by Neofusicoccum parvum has repeatedly been reported in recent years. The disease is a catastrophic disease of walnut with high incidence and is difficult to control. In order to establish a rapid and sensitive molecular detection system for N. parvum, two pairs of specific primers, CT-WK3-S / CT-WK3-A and CT-N-S / CT-N-A, were designed according to the sequence of chitin synthase 1 (CHS1) of N. parvum and 100 bp flanking sequences, and a nested PCR detection assay was established for N. parvum. The results showed that the established assay was specific to five strains of N. parvum from different sources and could amplify a 97 bp target with a sensitivity of 30 fg/μL, which was 10 times more sensitive than conventional PCR. No products were amplified in the related pathogenic species, Botryosphaeria sp., as well as the other tested strains. Therefore, the total DNA of walnut infected by N. parvum can be used as a template to detect the presence of N. parvum early in the disease. The nested PCR system established in this study can provide ideas for field detection of walnut branch dieback. |
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