洋葱黄矮病毒RPA-LFD可视化检测方法的建立及应用

Establishment and application of a visual detection method for onion yellow dwarf virus using RPA-LFD

中文关键词:洋葱黄矮病毒  重组酶聚合酶扩增-侧流层析试纸条  大蒜  快速检测

keywords:onion yellow dwarf virus  RPA-LFD  garlic  rapid detection

中文摘要:为建立洋葱黄矮病毒（onion yellow dwarf virus,OYDV）的田间快速检测方法，通过筛选特异性扩增引物、设计探针建立基于重组酶聚合酶扩增（recombinase polymerase amplification,RPA）和侧流层析试纸条（lateral flow dipstick,LFD）相结合的可视化检测方法，并优化该方法的反应温度和反应时间，验证特异性和灵敏度以及田间样品适用性。结果表明：所建RPA-LFD可视化检测方法有效反应温度范围为38～46℃，最适反应温度为44℃，最短反应时间为6 min。特异性和灵敏度结果显示，该方法对OYDV具有高度特异性，与韭葱黄条病毒（leek yellow stripe virus,LYSV）、大蒜潜隐病毒（garlic latent virus,GLV）和大蒜普通潜隐病毒（garlic common latent virus,GCLV）等9种常见葱属Allium作物病毒均无交叉反应；对OYDV外壳蛋白基因的检测下限可达10² copies/μL，灵敏度为聚合酶链反应（polymerase chain reaction,PCR）的10倍，且可稳定用于田间样品中OYDV的准确检测。该方法操作简便，无需精密仪器与专业操作技能，适用于田间现场快速检测。

Abstract:To establish a rapid field detection method for onion yellow dwarf virus(OYDV), this study developed a visual detection method based on recombinase polymerase amplification(RPA) combined with a lateral flow dipstick(LFD). Specific amplification primers were screened, probes designed, and the reaction temperature and reaction time optimized. The specificity, sensitivity, and field sample applicability of this method were verified, and an OYDV RPA-LFD visual detection system was successfully established. The results showed that the optimal primer-probe combination selected in this study enabled an effective RPA-LFD assay with a reaction temperature range of 38 ℃ to 46 ℃, an optimal reaction temperature of 44 ℃, and a minimum reaction time of six minutes. Specificity and sensitivity tests demonstrated that the method is highly specific to OYDV, showing no cross-reactivity with nine other common Allium-infecting viruses, including leek yellow stripe virus(LYSV), garlic latent virus(GLV), and garlic common latent virus(GCLV). The detection limit for the OYDV coat protein(CP) gene was 10² copies/μL, which is ten times more sensitive than polymerase chain reaction(PCR). Moreover, the assay reliably detected OYDV in field samples. It is easy to operate, requires no sophisticated instrument or professional expertise, and is suitable for rapid on-site detection, thus providing technical support for early monitoring and management of OYDV.