ISHS


Acta
Horticulturae
Home


Login
Logout
Status


Help

ISHS Home

ISHS Contact

Consultation
statistics
index


Search
 
ISHS Acta Horticulturae 722: XI International Symposium on Virus Diseases of Ornamental Plants

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

Authors:   R. Miglino, A. Jodlowska, A.R. van Schadewijk
Keywords:   RT-PCR, Allium, paramagnetic beads, ssRNA isolation, potexvirus group test
DOI:   10.17660/ActaHortic.2006.722.35
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 replicase–encoding 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.

Download Adobe Acrobat Reader (free software to read PDF files)

722_34     722     722_36

URL www.actahort.org      Hosted by KU Leuven      © ISHS