Conventional PCR Detection and Real-Time PCR Quantification of Reniform Nematodes
Conventional PCR Detection and Real-Time PCR Quantification of Reniform Nematodes
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DOI：
10.1094/pdis-12-11-1033-re
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发表时间：
2012-12-01
期刊：
影响因子：
4.5
通讯作者：
Kirkpatrick, Terrence
中科院分区：
文献类型：
--
作者：
Sayler, Ronald J.;Walker, Courtney;Kirkpatrick, Terrence
肾形线虫(Rotylichulus Reniformis)是一种较新引入美国大陆的线虫，可导致包括棉花和大豆在内的各种重要作物的重大产量损失。该线虫内转录间隔区(ITS)的DNA序列被用来设计用于常规和实时聚合酶链式反应的引物，以及TaqMan探针。这些引物扩增了来自广泛地理范围的肾形线虫分离物的DNA，但没有检测到遗传相关物种或生产领域中发现的其他致病线虫，包括南方根结线虫和大豆异源线虫。SYBR Green和TaqMan两种检测方法都可靠地定量了低至100fg的肾形线虫DNA，并可用于定量少至五种肾形线虫。描述了一种用于高通量诊断分析的廉价、快速的DNA提取方法。
Reniform nematode (Rotylenchulus reniformis) is a relatively recent introduction into the continental United States that can cause major yield losses on a variety of important crops including cotton and soybeans. DNA sequences from the internal transcribed spacer (ITS) region of this nematode were used to design primers for conventional and real-time PCR, as well as a TaqMan probe. These primers amplified DNA of reniform nematode isolates from a wide geographic range but did not detect genetically related species or other pathogenic nematodes found in production fields including Meloidogyne incognita and Heterodera glycines. Both SYBR green and TaqMan assays reliably quantified as little as 100 fg of reniform nematode DNA, and could be used to quantify as few as five reniform nematodes. An inexpensive and rapid DNA extraction protocol for high throughput diagnostic assays is described.