Sensitive non-radioactive nucleic acid hybridization assay for plum pox virus detection

A new non-radioactive sandwich hybridization assay was designed to simplify the analysis of a large number of plant samples. Plant material was homogenized in 0.5% SDS and added directly to the hybridization reaction, in which a pair of identifying probes were used. One of the probes was biotinylated capture RNA specific for plum pox virus (PPV) strain SK-68; the other RNA probe was synthesized from a plasmid bearing the adjacent sequence of this strain and was labelled with digoxigenin (DIG). Both purified viral RNA and crude extracts from PPV-infected plants were used as target for sandwich hybridization. The hybridization reaction was carried out in a streptavidin-coated ELISA plate. After extensive washing, the viral RNA was detected by conventional colour reaction using anti-DIG/alkaline phosphatase conjugate. In comparative experiments, we have shown that this non-radioactive detection system is more sensitive than conventional ELISA techniques and we were able to detect virus-specific RNA in more than 50% of the ELISA-negative samples.