MacKenzie Donald J, McLean Morven A, Mukerji Srima, Green Margaret
Centre for Plant Health, Agriculture and Agri-Food Canada, 8801 East Saanich Road, Sidney, BC Canada V8L 1H3.
Plant Dis. 1997 Feb;81(2):222-226. doi: 10.1094/PDIS.1997.81.2.222.
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.
本文描述了一种从木本植物中提取高质量RNA的有效方法，该方法无需使用苯酚、有机溶剂或乙醇沉淀。该方法采用市售的旋转柱基质，减轻了在将传统提取方法应用于木本植物物种时常见的植物多糖和多酚化合物对后续聚合酶链反应扩增的抑制作用。所描述的方法已成功用于开发高灵敏度逆转录-聚合酶链反应（RT-PCR）技术，以检测木本宿主中的多种病毒。检测到的病毒包括苹果茎沟病毒（ASGV）、苹果茎痘病毒、李坏死环斑病毒（PNRSV）、葡萄扇叶病毒和南芥菜花叶病毒，以及葡萄卷叶相关病毒3型。通过对这些组织中的ASGV和PNRSV进行等效的RT-PCR检测，发现所描述的方法对于从接穗、叶片或花朵中提取病毒RNA同样有效。使用该方法制备的植物总RNA样品中病毒RNA的检测灵敏度与先前描述的免疫捕获RT-PCR技术相同。
