A PCR membrane spot assay for the detection of plum pox virus RNA in bark of infected trees

A procedure for sensitive detection of plum pox virus RNA in infected bark of trees is described. The method is based on the extraction of bark material with buffer containing proteinase K followed by partial purification of RNA using QUIAGEN anion exchange resin. The RNA is then reverse transcribed, the single stranded cDNA is amplified by the polymerase chain reaction using biotinylated deoxynucleotides as label. The amplified cDNA can subsequently be detected by spotting the reaction mixture onto a nitrocellulose membrane. After fixation and washing the incorporated label is detected enzymatically using streptavidin-alkaline phosphatase. It was shown that this non-radioactive detection system is more sensitive than ELISA and a DNA/RNA hybridization test using 32P-labelled probes. It is also possible to detect plum pox virus infection with this assay in trees in the non-vegetative period.