A PCR-microplate hybridization method for plant virus detection.

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 x 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 Immunoplate 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 10,000 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.

Characterization and strain identification of a potato virus Y isolate non-reactive with monoclonal antibodies specific to the ordinary and necrotic strains.

An isolate of potato virus Y, named PVY-36, reacted with polyclonal antibody against PVY-O (an ordinary strain), but not with any of eight monoclonal antibodies (MAbs) specific to PVY-O or with two MAbs specific to PVY-T (a necrotic strain). From its host range and symptomatology, PVY-36 belongs to the PVYO group. The nucleotide sequence of the coat protein (CP) coding region of the PVY-36 genome was determined and the amino acid sequence was predicted. Based on the CP amino acid sequence, PVY-36 is more closely related to PVY-O than to PVY-T. There were eight amino acid differences between the CPs of PVY-36 and PVY-O in the N-terminal 30 amino acids. It is suggested that amino acids 8-15 and/or 26-30 from the N-terminus may determine a PVY-O-specific epitope (or epitopes).

Molecular cloning, sequencing, and expression in Escherichia coli of the potato virus Y cytoplasmic inclusion gene.

Complete nucleotide sequences of cytoplasmic inclusion (CI) genes of two strains of potato virus Y (PVY) were determined from six polymerase chain reaction (PCR)-amplified cDNA clones. The size of the CI genes of both ordinary (PVY-O) and necrotic strains (PVY-T13) was 1902 nucleotides, with a sequence homology of 83.4%. Comparison of the predicted amino acid sequences showed more than 90% homology. When these were compared with those of other potyviruses, the homology ranged from 53 to 61%. cDNAs of all or a part of the PVY-O CI gene containing an additional initiation codon (ATG) at the 5' end and a stop codon at the 3' end were constructed by PCR amplification and cloned into an Escherichia coli expression vector, pKK 223-3. Complete and truncated PVY-O CI proteins were successfully produced in E. coli as judged by reactivities with PVY-O CI protein-specific antiserum. To our knowledge, this is the first report on expression of PVY CI proteins in E. coli.

Production and characteristics of strain common antibodies against a synthetic polypeptide corresponding to the C-terminal region of potato virus Y coat protein.

Comparing the predicted amino acid sequence between two Japanese potato virus Y (PVY) strains, necrotic strain and ordinary strain, it was found that the C-terminal regions (H2N-HTTEDVSPSMHTLLGVKNM-COOH) of the coat proteins in the two strains were completely conserved. The conserved amino acid sequence was also found in the C-terminal region coat protein of PVY-36, a strain which did not react with monoclonal antibodies specific to the necrotic and the ordinary strain respectively. Antibodies were produced against a synthetic polypeptide PVY-C19 consisting of 19 amino acids, which correspond to the C-terminal region of the coat protein, using 4 coupling combinations of polypeptide PVY-C19 to protein carriers. Carrier-free polypeptides and those coupled to ovalbumin with ECDI (ethyl-dimethylaminopropyl carbodiimide) produced high titer of antibodies and detected PVY strains from PVY-infected plants by Western blot analysis and by ELISA.