Oregon State University, OR, USA.
Phytopathology. 2012 Jan;102(1):14-22. doi: 10.1094/PHYTO-03-11-0093.
The root-lesion nematode Pratylenchus thornei is one of the most important pests restricting productivity of wheat in the Pacific Northwest (PNW). It is laborious and difficult to use microscopy to count and identify the nematodes in soils. A SYBR Green I-based real-time polymerase chain reaction (PCR) assay was developed to detect and quantify this species from DNA extracts of soil. A primer set, designed from the internal transcribed spacer region (ITS1) of rDNA, was highly specific to P. thornei and did not amplify DNA from 27 isolates of other Pratylenchus spp., other nematodes, and six fungal species present in PNW wheat fields. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. The standard curve was supported by a high correlation between the numbers of P. thornei added to soil and the numbers quantified using real-time PCR. Examination of 15 PNW dryland field soils and 20 greenhouse samples revealed significant positive correlations between the numbers determined by real-time PCR and by the Whitehead tray and microscopic method. Real-time PCR is a rapid, sensitive alternative to time-consuming nematode extractions, microscopic identification, and counting of P. thornei from field and greenhouse soils.
根结线虫 Pratylenchus thornei 是限制太平洋西北地区（PNW）小麦生产力的最重要害虫之一。使用显微镜对土壤中的线虫进行计数和鉴定既费力又困难。本文开发了一种基于 SYBR Green I 的实时聚合酶链反应（PCR）检测方法，可从土壤 DNA 提取物中检测和定量该物种。从 rDNA 的内部转录间隔区（ITS1）设计的引物对对 P. thornei 具有高度特异性，不会扩增来自 PNW 小麦田中 27 个其他 Pratylenchus 种、其他线虫和 6 种真菌的 DNA。从人工侵染土壤中生成了一个与阈值循环和线虫数量对数相关的标准曲线。该标准曲线得到了通过实时 PCR 添加到土壤中的 P. thornei 数量与实际定量数量之间高度相关性的支持。对 15 个 PNW 旱地土壤和 20 个温室样本的检测表明，实时 PCR 确定的数量与 Whitehead 托盘和显微镜方法确定的数量之间存在显著正相关。实时 PCR 是一种替代费时费力的线虫提取、显微镜鉴定和从田间及温室土壤中对 P. thornei 进行计数的快速、敏感方法。
