基于环介导等温扩增技术检测Colletotrichum camelliae

doi:10.13305/j.cnki.jts.2025.05.007

摘要/Abstract

摘要： 山茶炭疽菌Colletotrichum camelliae是引起茶炭疽病的优势病原菌,该病害可致茶树大量发病叶片脱落,严重影响茶叶产量,造成较大的经济损失。因此,早期及时检测茶树叶片中有无C. camelliae侵染,对生产上及时防治茶炭疽病有重要意义。基于环介导等温扩增（Loop-mediated isothermal amplification,LAMP）技术,以C. camelliae中1个编码LEA（Late embryogenesis abundant）结构域蛋白的特异基因作为靶标,设计并筛选了3对特异性LAMP引物,包括1对外引物、1对内引物和1对环引物。随后通过特异性和灵敏度检测,以及室内接种和田间发病组织检测试验,建立了C. camelliae的LAMP检测方法。该方法在65 ℃下反应30 min效果最佳,检测结果可通过2%琼脂糖凝胶电泳验证和SYBR Green Ⅰ显色判断。在特异性检测试验中,仅C. camelliae扩增后呈阳性反应;灵敏度检测试验中,该方法对C. camelliae基因组DNA模板的最低检测质量浓度为100 ag·µL^-1;对室内接种C. camelliae后的病叶和田间18个茶树品系的炭疽病病叶的检测结果显示,该LAMP方法能准确地检测出C. camelliae。综上,本研究建立了1种C. camelliae的LAMP检测方法,具有快速简便、灵敏度高等优点,可实现C. camelliae的特异性检测及茶炭疽病的快速诊断。

关键词: 茶炭疽病, 山茶炭疽菌, 环介导等温扩增技术, 病害检测, 特异基因

Abstract: Colletotrichum camelliae is the dominant pathogen causing tea anthracnose. This disease causes significant defoliation, which has a major impact on the following year's yield and results in great economic losses. Therefore, the early and timely detection of C. camelliae in tea leaves is of great significance for effective control of anthracnose in production. In this study, based on a loop-mediated isothermal amplification (LAMP) assay, a specific gene encoding a late embryogenesis abundant (LEA) domain protein from C. camelliae was selected for the design of LAMP primers. Three pairs of specific LAMP primers, including two outer primers, two inner primers, and two loop primers, were designed and selected. Subsequently, a LAMP assay for C. camelliae was established through specificity and sensitivity tests, and by detecting C. camelliae in artificially inoculated and infected tea leaves. The optimal reaction condition was determined to be at 65 ℃ for 30 min. The results were verified by 2% agarose gel electrophoresis with SYBR Green Ⅰ added. Specificity tests reveal that, only DNA extracted from C. camelliae isolates exhibited a positive amplification reaction. In sensitivity tests, the assay could detect genomic DNA templates of C. camelliae at a minimum concentration of 100 ag·µL^-1. The LAMP assay accurately detected C. camelliae in artificially inoculated diseased leaves and anthracnose-affected leaves of 18 tea germplasms in the field. In summary, the LAMP assay for detecting C. camelliae established in this study has the advantages of rapid, simple and high sensitivity, enabling the specific detection of C. camelliae and the quick diagnosis of tea anthracnose.

Key words: tea anthracnose, Colletotrichum camelliae, loop-mediated isothermal amplification, disease detection, specific gene

中图分类号:

涂一怡, 张幼, 徐婷, 陈俊杰, 王玉春, 吕务云. 基于环介导等温扩增技术检测Colletotrichum camelliae[J]. 茶叶科学, 2025, 45(5): 770-782. doi: 10.13305/j.cnki.jts.2025.05.007.

TU Yiyi, ZHANG You, XU Ting, CHEN Junjie, WANG Yuchun, LÜ Wuyun. Loop-mediated Isothermal Amplification-based Detection of Colletotrichum camelliae[J]. Journal of Tea Science, 2025, 45(5): 770-782. doi: 10.13305/j.cnki.jts.2025.05.007.

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