A simplified method for obtaining plant viral RNA for RT-PCR
A simplified method for obtaining plant viral RNA for RT-PCR
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DOI：
10.1016/j.jviromet.2005.01.002
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发表时间：
2005-04-01
影响因子：
3.1
通讯作者：
Natsuaki, T
中科院分区：
文献类型：
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作者：
Suehiro, N;Matsuda, K;Natsuaki, T
建立了一种简便、快速的植物病毒RNA制备方法（SDT法）。SDT方法可在约15分钟内完成，不需要使用抗血清、过滤或离心。在含有吐温-20（PBST）的磷酸盐缓冲盐水中研磨植物组织并将提取物置于微量离心管中几分钟的程序允许病毒颗粒吸附到管壁上。然后除去树液，并在加入无RNA酶的水之前用PBST洗涤管。该操作可在室温下进行。利用该方法，然后逆转录-聚合酶链反应（RT-PCR），感染萝卜花叶病毒，黄瓜花叶病毒，黄瓜绿色斑驳花叶病毒（CGMMV）很容易检测，表明SDT方法可以用于检测不同的病毒。为了检测CGMMV，在cDNA合成之前加热管是必要的，这表明固定化的CGMMV颗粒需要通过热处理来破坏以释放RNA。(c)2005 Elsevier B. V.保留所有权利。
An easy and fast procedure (named the simple-direct-tube (SDT) method) was developed for preparing plant virus RNA for cDNA synthesis. The SDT method can be completed in approximately 15 min and does not require the use of antiserum, filtering or centrifugation. The procedure to grind plant tissues in phosphate-buffered saline containing Tween-20 (PBST) and to place the extract in a microfuge tube for a few minutes allow adsorption of the virus particles to the tube wall. The sap is then removed and the tube is washed with PBST before the addition of RNase-free water. This manipulation can be performed at room temperature. Using this method followed by reverse transcription-polymerase chain reaction (RT-PCR), infections by turnip mosaic virus, cucumber mosaic virus, and cucumber green mottle mosaic virus (CGMMV) were readily detected, indicating that the SDT method can be used in assays to detect different viruses. For the detection of CGMMV, it was necessary to heat the tubes before cDNA synthesis, suggesting that the immobilized CGMMV particles required disruption by heat treatment to release RNA. (c) 2005 Elsevier B.V. All rights reserved.