A SINGLE TEMPERATURE AMPLIFICATION TECHNIQUE APPLIED TO THE DETECTIONOF 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 le aves and bark is described. The method requires the isolation and prec ipitation of total nucleic acids from either infected whole leaf or ba rk tissue. The CTV viral RNA is then specifically amplified using a si ngle temperature RNA Self-Sustained Sequence Replication technique (3S R(TM)) performed at 42 degrees C for 60 minutes. The amplified negativ e-sense viral RNA product can subsequently be detected by fixing a por tion of the reaction mixture onto a nylon membrane and hybridizing wit h positive sense tristeza specific DNA oligonucleotide probes. Central California isolates of CTV were readily detected by this method. Dena tured viral specific dsRNA was also a suitable template for the specif ic detection of CTV.