THE USE OF SHORT AND LONG PCR PRODUCTS FOR IMPROVED DETECTION OF PRUNUS NECROTIC RINGSPOT VIRUS IN WOODY-PLANTS

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of prunus necrotic ringspot virus (PNRSV) in dormant pea ch and almond trees by the application of two different pairs of prime rs yielding a short and a long product, respectively. The relative amo unt of the short (200 base pair, bp) product was higher than the longe r (785 bp) product. PNRSV was detected better in plant tissues with a low virus concentration (e.g. dormant trees) by amplification of the s hort PCR product, whereas the long product was produced at higher viru s titers. Simultaneous amplification of both short and long products w as demonstrated using a three-primer mixture in a single reaction tube . In this assay, amplification of either PCR product indicated the pre sence of PNRSV-specific sequences in the plant tissue examined, thus c overing a wide range of virus concentrations in a single test. Dilutio n of the RNA extracted from infected plant material resulted in a stee p decline in the amplification of both short and long PCR products. In contrast, serial dilutions of the intermediate cDNA template differen tially affected the amplification patterns: the relative amount of the short product increased whereas that of the long product decreased. T hese results may explain the preferential amplification of the short P CR product observed in samples containing low virus concentrations. (C ) 1997 Elsevier Science B.V.