基于CEEA等温扩增技术的落叶枯梢病菌快速检测技术体系

Establishment of a rapid detection technology system for Neofusicoccum laricinum based on CEEA isothermal amplification

摘要

落叶松枯梢病菌 Neofusicoccum laricinum (Sawada) Y. Hattori & C. Nakash 是中国进境植物检疫性有害生物和林业检疫性有害生物,也是《重点管理外来入侵物种名录》中仅有的2种真菌性植物病菌之一。开展快速、准确、直观的检测技术研究,对落叶松枯梢病的有效防治具有重要意义。本研究将复合酶指数扩增技术(CEEA)与侧向流试纸条(LFD)技术相结合,利用落叶松枯梢病菌的特异性CEEA引物组及其探针,在最佳反应温度39 ℃、反应时间15 min的条件下,特异性检测出不同地区来源的落叶松枯梢病菌,最低检测浓度为100.0 fg·µL⁻¹,且整个检测过程可在30 min内完成。同时,该技术无需昂贵仪器,且不识别落叶松上的其他真菌,表现出良好的适用性,具有高效、快速、灵敏等特点,可为林间病原菌的就地快速检测、识别以及病害早期预警提供有力支持。

Abstract

Neofusicoccum laricinum (Sawada) Y. Hattori & C. Nakash is a quarantine pest of imported plants and a forestry quarantine pest in China. It is also one of only two fungal plant pathogens listed in the Catalogue of Key Managed Invasive Alien Species. Developing rapid,accurate,and visual detection method is crucial for effective prevention and control of this disease. In this study,a method combining composite enzyme index amplification (CEEA) technique with lateral flow dipstick (LFD) technology was developed. Using specifically designed CEEA primer sets and probes for N. laricinum,followed by optimization of reaction conditions (39 ℃ for 15 minutes),the method could specifically detect N. laricinum strains from diverse regions with a limit of detection (LOD) as low as 100.0 fg·µL⁻¹,the entire process to be completed in 30 minutes. This technology requires no expensive instruments and does not identify other fungi on Larch,demonstrating good applicability. With its high efficiency,speed,and sensitivity,this technology provides strong support for in-situ rapid detection and identification of pathogens in the forest,as well as for the early warning of disease.

关键词

新壳梭孢菌 / 落叶松 / 复合酶指数扩增 / 侧向流试纸条 / 检测技术

Key words

Neofusicoccum laricinum / Larch / composite enzyme index amplification / lateral flow test strips / detection technology

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基金资助

国家重点研发计划(2021YFD1400300)