Articles
DETECTION OF CHRYSANTHEMUM STUNT VIROID BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION AND BY TISSUE BLOT HYBRIDIZATION
Article number
432_15
Pages
120 – 129
Language
Abstract
Reverse transcription-polymerase chain reaction (RT-PCR) assays were developed for the detection and identification of chrysanthemum stunt viroid (CSVd) from sap, or total nucleic acid extracts of infected dried or freshly collected chrysanthemum leaves.
DNA primers specific for CSVd sequence were used for cDNA synthesis and specific amplification of CSVd.
The antisense primer was complementary to CSVd nucleotide 68–87 in the central conserved region and the sense primer was homologous to CSVd nucleotide 88–112. A full length CSVd cDNA 356 bp product was detected from infected but not from uninfected tissue.
This product hybridized specifically to digoxigenin-labeled CSVd cRNA probe.
CSVd cRNA was detected from 100 ng to as little as 0.1 ng of total initial nucleic acids from infected tissue.
The amount of amplified CSVd cDNA decreased as total initial nucleic acids decreased.
Similarly, a 356 bp CSVd cDNA product was detected from clarified sap extracts of infected tissue diluted 100 to 1000 fold.
Treatment of diluted extracts with GeneReleaser™ polymeric matrix to bind to RT-PCR inhibitors may increase the sensitivity of the assay.
Tissue blot of cross-sectioned CSVd-infected chrysanthemum stems or leaf petioles gave positive reactions when hybridized with the digoxigenin-labeled probe.
However, stronger hybridization signals were obtained from leaf petioles than from stems.
No reaction with similar tissues from healthy plants was observed.
DNA primers specific for CSVd sequence were used for cDNA synthesis and specific amplification of CSVd.
The antisense primer was complementary to CSVd nucleotide 68–87 in the central conserved region and the sense primer was homologous to CSVd nucleotide 88–112. A full length CSVd cDNA 356 bp product was detected from infected but not from uninfected tissue.
This product hybridized specifically to digoxigenin-labeled CSVd cRNA probe.
CSVd cRNA was detected from 100 ng to as little as 0.1 ng of total initial nucleic acids from infected tissue.
The amount of amplified CSVd cDNA decreased as total initial nucleic acids decreased.
Similarly, a 356 bp CSVd cDNA product was detected from clarified sap extracts of infected tissue diluted 100 to 1000 fold.
Treatment of diluted extracts with GeneReleaser™ polymeric matrix to bind to RT-PCR inhibitors may increase the sensitivity of the assay.
Tissue blot of cross-sectioned CSVd-infected chrysanthemum stems or leaf petioles gave positive reactions when hybridized with the digoxigenin-labeled probe.
However, stronger hybridization signals were obtained from leaf petioles than from stems.
No reaction with similar tissues from healthy plants was observed.
Authors
R. Hooftman, M.-J. Arts, A.M. Shamloul, A. van Zaayen, A. Hadidi
Keywords
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