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 peach and almond trees by the application of two different pairs of primers yielding a short and a long product, respectively. The relative amount of the short (200 base pair, bp) product was higher than the longer (785 bp) product. PNRSV was detected better in plant tissues with a low virus concentration (e.g. dormant trees) by amplification of the short PCR product, whereas the long product was product was produced at higher virus titers. Simultaneous amplification of both short and long products was demonstrated using a three-primer mixture in a single reaction tube. In this assay, amplification of either PCR product indicated the presence of PNRSV-specific sequences in the plant tissue examined, thus covering a wide range of virus concentrations in a single test. Dilution of the RNA extracted from infected plant material resulted in a steep decline in the amplification of both short and long PCR products. In contrast, serial dilutions of the intermediate cDNA template differentially affected the amplification patterns: the relative amount of the short product increased whereas that of the long product decreased. These results may explain the preferential amplification of the short PCR product observed in samples containing low virus concentrations.
Rosner
A
A
Department of Virology, Volcani Center, Bet Dagan, Israel. VPSARASP@VOLCANI.AGRI.GOV.IL
Maslenin
L
L
Spiegel
S
S
D018076:DNA, Complementary / Q000737:chemistry; D005638:Fruit; D019179:Ilarvirus / Q000737:chemistry / Q000302:isolation & purification*; D009754:Nuts; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase; D013698:Templates, Genetic; D014197:Trees / Q000821:virology
D018076:DNA, Complementary; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase