Thomson D, Dietzgen R G
Department of Primary Industries, Queensland Agricultural Biotechnology Centre, Gehrmann Laboratories, University of Queensland, Brisbane, Australia.
J Virol Methods. 1995 Aug;54(2-3):85-95. doi: 10.1016/0166-0934(95)00022-m.
A simple, single-step plant tissue preparation protocol suitable for the detection of viruses by the polymerase chain reaction and reverse transcription-polymerase chain reaction is described. The effect of buffer components and pH, and the incubation temperature for the release of virus from plant material was evaluated. A small amount of plant tissue was heated in a solution containing 100 mM Tris-HCl, pH 7.4 or 8.4, 1 M KCl and 10 mM EDTA for 10 min at 95 degrees C and the supernatant used for enzymatic amplification. This protocol was suitable for the detection of both DNA and RNA viruses in a variety of plant species and tissues and reduced plant inhibitory factors which may interfere with PCR. The application of this method was demonstrated for the detection of banana bunchy top virus in banana leaves, root and corn, zucchini yellow mosaic potyvirus in squash leaves and lettuce necrotic yellows rhabdovirus in lettuce and Nicotiana glutinosa leaves.
描述了一种简单的单步植物组织制备方案，适用于通过聚合酶链反应和逆转录 - 聚合酶链反应检测病毒。评估了缓冲液成分和pH值以及从植物材料中释放病毒的孵育温度的影响。将少量植物组织在含有100 mM Tris-HCl（pH 7.4或8.4）、1 M KCl和10 mM EDTA的溶液中于95℃加热10分钟，然后将上清液用于酶促扩增。该方案适用于检测多种植物物种和组织中的DNA和RNA病毒，并减少了可能干扰PCR的植物抑制因子。该方法的应用已在香蕉叶、根和玉米中检测香蕉束顶病毒、南瓜叶中检测西葫芦黄花叶马铃薯Y病毒以及生菜和烟草叶中检测生菜坏死黄化弹状病毒中得到证明。
