ISHS Acta Horticulturae 656: X International Symposium on Small Fruit Virus Diseases
DIAGNOSIS AND QUANTIFICATION OF STRAWBERRY VEIN BANDING VIRUS USING MOLECULAR APPROACHES |
| Author: | A. Mahmoudpour |
| Keywords: | Limits of PCR inhibition/detection; nucleic acid extraction; virus diagnosis |
| Abstract:
PCR and gel analyses were used for detection and quantification of Strawberry vein banding virus. A viral DNA isolation procedure was optimized to allow quick isolation of total nucleic acids prior to PCR. Different aged leaves, petioles, flowers and roots from infected strawberry plants including a virus indicator and three commonly grown cultivars were used to determine the most suitable tissues for virus testing and evaluate different diagnostic procedures. Nucleic acid preparations were diluted 10-10,000 fold in sterile water to determine the detection limits of PCR and the relative virus titer of samples based on dilution end point assay. The highest virus titer was in old symptomatic leaves and the lowest was in petioles. However, virus titer varied greatly among source plants used. To determine the PCR inhibition limits, concentrated preparations (up to 100x higher than those used in routine diagnosis) were used as templates. False negative results were interpreted as inhibition of Taq DNA polymerase activity by host derived contaminants with the highest levels in terminal roots and the lowest in young terminal leaves. These data were used to design a framework in mass screening field samples by pooling multiple samples. Colorimetric PCR and dot blot hybridization assay were used to confirm the above findings. | |
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