Articles
DETECTION AND IDENTIFICATION OF A NOVEL POTEXVIRUS INFECTING ALLIUM BY PARAMAGNETIC BEADS SSRNA ISOLATION AND ONE TUBE RT-PCR ASSAY WITH A NEW POTEXVIRUS GENUS PRIMER SET
Article number
722_35
Pages
285 – 292
Language
English
Abstract
A rapid paramagnetic capture/reverse transcription PCR assay procedure was developed for the detection of viruses from the genus Potexvirus.
The Potexvirus ssRNA was selectively isolated from plants sap by streptavidin coated paramagnetic beads coupled to a biotin labelled Potexvirus genus sequence specific primer/probe and then analysed in a one tube RT-PCR assay.
An extraction buffer containing cell wall degrading enzymes was used in order to improve the viral detection in bulbs.
The RT-PCR assay was performed using a new Potexvirus genus specific primer set.
Upstream primer Potex 4 and downstream primer Potex 5 were developed on the sequence of conserved viral replicaseencoding regions.
Ornamental Allium plants showing diffuse yellow stripes or slight mottle were investigated for virus infection.
ELISA and PCR assay showed the presence of a complex of different viruses consisting of Potyviruses, Tobacco rattle virus, Tobacco necrosis virus and a novel (unknown) Potexvirus. Sequence analysis of cloned PCR amplicon obtained with the primers Potex 4 Potex 5, showed an 83% identity with Clover yellow mosaic virus.
Testing the method on primarily infected plants and dormant bulbs of different Allium cultivars has verified the robustness for application in the routine detection of potexvirus infections.
Detection, performed in microtiter plates, was rapid, specific, contamination free, reproducible and semi-automated.
The Potexvirus ssRNA was selectively isolated from plants sap by streptavidin coated paramagnetic beads coupled to a biotin labelled Potexvirus genus sequence specific primer/probe and then analysed in a one tube RT-PCR assay.
An extraction buffer containing cell wall degrading enzymes was used in order to improve the viral detection in bulbs.
The RT-PCR assay was performed using a new Potexvirus genus specific primer set.
Upstream primer Potex 4 and downstream primer Potex 5 were developed on the sequence of conserved viral replicaseencoding regions.
Ornamental Allium plants showing diffuse yellow stripes or slight mottle were investigated for virus infection.
ELISA and PCR assay showed the presence of a complex of different viruses consisting of Potyviruses, Tobacco rattle virus, Tobacco necrosis virus and a novel (unknown) Potexvirus. Sequence analysis of cloned PCR amplicon obtained with the primers Potex 4 Potex 5, showed an 83% identity with Clover yellow mosaic virus.
Testing the method on primarily infected plants and dormant bulbs of different Allium cultivars has verified the robustness for application in the routine detection of potexvirus infections.
Detection, performed in microtiter plates, was rapid, specific, contamination free, reproducible and semi-automated.
Authors
R. Miglino, A. Jodlowska, A.R. van Schadewijk
Keywords
RT-PCR, Allium, paramagnetic beads, ssRNA isolation, potexvirus group test
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