Improved RNA extraction from woody plants for the detection of viral pathogens by reverse transcription-polymerase chain reaction
Improved RNA extraction from woody plants for the detection of viral pathogens by reverse transcription-polymerase chain reaction
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DOI：
10.1094/pdis.1997.81.2.222
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发表时间：
1997-02-01
期刊：
影响因子：
4.5
通讯作者：
Green, M
中科院分区：
文献类型：
--
作者：
MacKenzie, DJ;McLean, MA;Green, M
本文描述了一种从木本植物中提取高质量RNA的有效方法，该方法不使用酚、有机溶剂或醇沉淀。该方法采用市售的旋转柱矩阵和减轻抑制作用的植物多糖和多酚化合物通常观察到的后续聚合酶链反应扩增时，常规提取方法应用于木本植物物种。所描述的方法已成功地用于开发高灵敏度的逆转录-聚合酶链反应（RT-PCR）技术，用于检测其木本宿主中的一些病毒。检测到的病毒包括苹果茎沟毛细病毒（ASGV），苹果茎痘病毒，李坏死环斑病毒（PNRSV），葡萄藤扇叶和南芥花叶线虫传多角体病毒，和葡萄藤卷叶相关的线形病毒3型。通过对这些组织中的ASGV和PNRSV进行等效RT-PCR检测，确定所描述的方法对于从芽木、叶子或花朵中提取病毒RNA同样有效。发现使用该方法制备的总植物RNA样品中的病毒RNA检测与先前描述的免疫捕获RT-PCR技术一样灵敏。
An efficient procedure for the extraction of high-quality RNA from woody plants without the use of phenol, organic solvents, or alcohol precipitation is described. The method employs commercially available spin-column matrices and mitigates the inhibitory effects of plant polysaccharides and polyphenolic compounds commonly observed on subsequent polymerase chain reaction amplification when conventional extraction methods are applied to woody plant species. The method described has been successfully used in the development of highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) techniques for the detection of a number of viruses in their woody hosts. The viruses detected included apple stem grooving capillovirus (ASGV), apple stem pitting virus, Prunus necrotic ringspot ilarvirus (PNRSV), grapevine fanleaf and Arabis mosaic nepoviruses, and grapevine leafroll-associated closterovirus type 3. The method described was equally effective for the extraction of viral RNA from either budwood, leaves, or flower blossoms as determined by the equivalent RT-PCR detection of ASGV and PNRSV from these tissues. Detection of viral RNA in samples of total plant RNA prepared using this method was found to be as sensitive as was previously described for the immunocapture RT-PCR technique.