A single temperature amplification technique applied to the detection of citrus tristeza viral RNA in plant nucleic acid extracts.

A procedure for the successful detection of citrus tristeza virus (CTV) RNA in total crude nucleic acid extracts of infected citrus whole leaves and bark is described. The method requires the isolation and precipitation of total nucleic acids from either infected whole leaf or bark tissue. The CTV viral RNA is then specifically amplified using a single temperature RNA Self-Sustained Sequence Replication technique (3SR) performed at 42 degrees C for 60 minutes. The amplified negative-sense viral RNA product can subsequently be detected by fixing a portion of the reaction mixture onto a nylon membrane and hybridizing with positive sense tristeza specific DNA oligonucleotide probes. Central California isolates of CTV were readily detected by this method. Denatured viral specific dsRNA was also a suitable template for the specific detection of CTV.