ISHS Acta Horticulturae 386: XVI International Symposium on Fruit Tree Virus diseases
AN IMMUNOCAPTURE PCR ASSAY ADAPTED TO THE DETECTION AND THE ANALYSIS OF THE MOLECULAR VARIABILITY OF APPLE CHLOROTIC LEAF SPOT VIRUS |
| Authors: | T. Candresse, M. Lanneau, F. Revers, G. Macquaire, S. German, J. Dunez, N. Grasseau, T. Malinovsky |
| DOI: | 10.17660/ActaHortic.1995.386.17 |
| Abstract:
We have developed a sensitive and polyvalent PCR-based assay for the detection of apple chlorotic leaf spot virus (ACLSV). To achieve maximal sensitivity, an immunocapture (IC) step is carried out directly in the PCR tube before the reverse transcription and PCR reactions. In its optimized form, the assay allows detection of 10 fg (1 fg = 10-12 g) of purified virus, which is equivalent to about 100 virus particles. The assay has proved very useful for the detection of ACLSV in a variety of plant material (leaves but also bark, flowers…) and from a variety of woody hosts, including apple, peach, cherry, apricot etc. The results of a full year indexing trial with both ELISA and IC-PCR demonstrate the increased rate of ACLSV detection with IC-PCR, especially during the summer period. The IC-PCR assay was also used, in conjunction with sequencing of the amplified fragment, to study the molecular variability of ACLSV. Extensive sequence divergence between ACLSV isolates is observed: homology levels of randomly selected isolates are usually in the 80–90% range. Most coding differences were observed in the putative viral movement protein while the coat protein showed better conservation, in keeping with the already known limited serological variability of ACLSV. | |
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