Poudel Dinesh, Yan Guiping
Department of Plant Pathology, North Dakota State University, Fargo, ND 58108, USA.
Int J Mol Sci. 2025 Aug 9;26(16):7711. doi: 10.3390/ijms26167711.
The root-lesion nematode, , is a migratory endoparasite that attacks potato roots, causing necrotic lesions and yield losses of up to 73%. Traditional detection and quantification methods are labor-intensive, time-consuming, and require prior nematode extraction and taxonomic expertise. This study aimed to develop a SYBR Green-based real-time quantitative PCR (qPCR) assay for detecting and quantifying directly from potato root DNA extracts. Bovine serum albumin (BSA) was tested to neutralize qPCR inhibitors in root DNA extracts. The assay showed high specificity and sensitivity to , detecting 1.56 × 10 of a single nematode in 0.2 g of roots. A standard curve based on artificial nematode inoculations demonstrated a strong linear relationship between Cq values and log-transformed nematode numbers ( = 0.993) with high amplification efficiency. Assessment using root samples from two greenhouse experiments involving five potato cultivars showed strong correlations ( = 0.902 and 0.887) between qPCR estimates and microscopic nematode counts. This study presents a new qPCR assay specifically optimized for direct detection and quantification of within potato root tissues, offering enhanced sensitivity and applicability for rapid in planta diagnostics to facilitate effective management strategies.
根结线虫是一种迁移性内寄生线虫，会侵袭马铃薯根系，导致坏死斑，产量损失高达73%。传统的检测和定量方法劳动强度大、耗时，且需要事先进行线虫提取和分类学专业知识。本研究旨在开发一种基于SYBR Green的实时定量PCR（qPCR）检测方法，用于直接从马铃薯根DNA提取物中检测和定量。测试了牛血清白蛋白（BSA）以中和根DNA提取物中的qPCR抑制剂。该检测方法对根结线虫显示出高特异性和高灵敏度，在0.2克根中能检测到1.56×10个单条线虫。基于人工接种线虫的标准曲线表明，Cq值与对数转换后的线虫数量之间存在很强的线性关系（=0.993），扩增效率高。使用来自涉及五个马铃薯品种的两个温室实验的根样本进行评估表明，qPCR估计值与显微镜下的线虫计数之间存在很强的相关性（=0.902和0.887）。本研究提出了一种专门针对马铃薯根组织中根结线虫的直接检测和定量进行优化的新qPCR检测方法，为植物体内快速诊断提供了更高的灵敏度和适用性，以促进有效的管理策略。
