A PCR-microplate hybridization method for plant virus detection

Abstract

A sensitive nonradioactive method for detection of plant viruses was evaluated. A cDNA fragment from the coat protein coding region of the potato virus Y (PVY) RNA genome amplified by reverse transcription (RT) followed by polymerase chain reaction (PCR), was directly adsorbed onto polystyrene microplate wells after heat denaturation. The adsorbed cDNA was hybridized with a digoxigenin (DIG)-labelled cDNA probe without a prehybridization step. The probe was also prepared by PCR using the same pair of primers to amplify the target sequence from a cDNA clone of part of the viral genome. The hybrid of the adsorbed cDNA and DIG-labelled probe was reacted with alkaline phosphatase-conjugated anti-DIG antibody. The enzyme activity was then detected by hydrolysis of a substrate, and the absorbance values were measured using a microplate reader. Highest absorbance values were obtained when the amplified DNA fragments were diluted 100 or 125-fold in 10 × SSC (1.5 M NaCl, 0.15 M sodium citrate) for adsorption. Highly concentrated DNA gave lower absorbance values. The absorbance values differed depending on the microplates used, and the highest value was obtained using Nunc Immunopiate II-Maxisorp microplates. DNA fragments longer than 300 bp all gave similar absorbance values, which were twice as high as those obtained with shorter fragments. When the amplified DNA was diluted 100-fold, 10 fg of PVY genomic RNA could be detected by this method, which is called PCR-microplate hybridization. This is about 10000 times more sensitive than enzyme-linked immunosorbent assay (ELISA). By this method, PVY was detected from five field potato samples showing to be free from PVY by ELISA. PCR-microplate hybridization is nucleic acid-based ELISA-like highly sensitive diagnostic method, and may be generally applicable for detection of plant viruses, viroids, and possibly other plant pathogens.