Molecular tools
Detection and characterization of molecular identities of PVY isolates
Genetic differences between viral genomes can be used as specific targets for molecular detection and identification of PVY isolates. A common feature of any molecularly-based detection tool corresponds to specific hybridization of probes and/or primers on the targeted nucleotide sequence(s). Such hybridization steps can be used to detect the presence of the target(s) (e.g. radiolabelled primers/probes, Southern or northern blots, DNA chips) or to amplify (RT-PCR) part of the targeted nucleic acids. Then, the amplified fragments can be observed on agarose gel under UV irradiations in the presence of ethidium bromide.
For few years, fluorescent-based techniques have been developed for PVY detection. Such methods have improved the sensitivity and/or specificity of previously published molecular tools against this plant virus. The recent identification of PVY molecular determinant linked to the necrosis ability of some viral isolates has offered, in association with previously published neutral markers, the possibility to set up new series of innovative molecular tools for the detection (FRIT’N SNP test), the quantification (FRIT’N real time assays) and the characterization (FRIT’N SNaPshot test) of PVY isolates belonging to the different groups and variants. These FRIT’N tests will be extensively used by the “PVYwide Organization” to analyse and characterize the world-wide collection of PVY isolates.
The qualitative “FRIT’N SNP” detection uses a single nucleotide polymorphism to identify in tested samples isolates belonging to the PVYN or PVYO groups.
|
.png)
   |
.png)
|
The “FRIT’N Real time” quantification procedures require the use of two independent assays for the specific quantification of PVYN and PVYO isolates.
|
The “FRIT’N SNaPshot” characterization of PVY isolates and their assignment in the PVY groups (PVYN and PVYO) and variants (PVYN-W and PVYNTN) depends on the identity of four polymorphic nucleotides.
|
 |
Writing:
Agnès Delaunay
Creation date: 04 October 2007
Update: 02 January 2007
