ISHS Acta Horticulturae 193: XIII International Symposium on Fruit Tree Virus Diseases
SYNTHESIS AND CHARACTERIZATION OF A DNA COMPLEMENTARY TO PLUM POX VIRUS RNA TO BE USED AS A SPECIFIC DETECTION PROBE |
| Authors: | C. Varveri, C. Bretout, J. Dunez |
| Abstract:
Plum pox virus (PPV) RNA was extracted from purified virus suspensions. After polyacrylamide gel electrophoresis, PPV RNA appears as homogeneous molecules of 3.5 x 106d consisting of about 11 kb. These molecules were used as a template to be transcribed by AMV reverse transcriptase with an oligo dT primer. Transcription medium contained dithiothreitol, RNAsin, actinomycin D and 32p dCTP as labelled nucleotide. The size of transcripts was estimated by alkaline gel electrophoresis using Hind III fragments of bacteriophage DNA as markers. PPV cDNA consists of single-stranded molecules, as demonstrated by their sensitivity to S1 nuclease treatment, with sizes in the range of 5–11 kb : some are almost full length. Hybridization conditions have been determined. At 50°C and in the presence of 70 % formamide, the transcribed cDNA clearly hybridized with homologous purified PPV RNA and not with heterologous viral RNAs. When checked against total nucleic acid from plant extracts, PPV cDNA hybridized with preparations from infected plants and not with those from healthy plants, demonstrating the possibility of using such a cDNA as a probe for further detection of PPV in infected plants. | |
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