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Detection of DNA and RNA plant viruses by PCR and RT-PCR using a rapid virus release protocol without tissue
1Department of Primary Industries, Queensland Agricultural Biotechnology Centre, Gehrmann Laboratories, University of Queensland, Brisbane, Australia.
Abstract:
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.