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.
Palkovics
L
L
Agricultural Biotechnology Center, Hungary.
Burgyán
J
J
Balázs
E
E
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D009693:Nucleic Acid Hybridization; D017801:Plum Pox Virus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis*; D012680:Sensitivity and Specificity
D012367:RNA, Viral