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doi:10.1016/j.jviromet.2007.08.019 
Copyright © 2007 Elsevier B.V. All rights reserved.
Oligonucleotide microarray-based detection and genotyping of Plum pox virus
Graziella Pasquinia, Marina Barbaa, 
, 
, Ahmed Hadidib, 1, Francesco Faggiolia, Rodolfo Negric, Iris Sobold, Antonio Tiberinia, Kadriye Caglayane, Hamed Mazyadf, Ghandi Anfokag, Murad Ghanimh, Mohammad Zeidani and Henryk Czosnekd
Received 16 May 2007;
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Abstract
Plum pox virus (PPV) is the most damaging viral pathogen of stone fruits. The detection and identification of its strains are therefore of critical importance to plant quarantine and certification programs. Existing methods to screen strains of PPV suffer from significant limitations such as the simultaneous detection and genotyping of several strains of PPV in samples infected with different isolates of the virus.
A genomic strategy for PPV screening based on the viral nucleotide sequence was developed to enable the detection and genotyping of the virus from infected plant tissue or biological samples. The basis of this approach is a long 70-mer oligonucleotide DNA microarray capable of simultaneously detecting and genotyping PPV strains. Several 70-mer oligonucleotide probes were specific for the detection and genotyping of individual PPV isolates to their strains. Other probes were specific for the detection and identification of two or three PPV strains. One probe (universal), derived from the genome highly conserved 3′ non-translated region, detected all individual strains of PPV. This universal PPV probe, combined with probes specific for each known strain, could be used for new PPV strain discovery. Finally, indirect fluorescent labeling of cDNA with cyanine after cDNA synthesis enhanced the sensitivity of the virus detection without the use of the PCR amplification step.
The PPV microarray detected and identified efficiently the PPV strains in PPV-infected peach, apricot and Nicotiana benthamiana leaves. This PPV detection method is versatile, and enables the simultaneous detection of plant pathogens.
Keywords: Diagnosis; Genotyping; Microarrays; 70-mer oligonucleotide probe; Plum pox virus; PPV strains; PPV-D; PPV-M; PPV-C; PPV-EA
Article Outline
- 1. Introduction
- 2. Materials and methods
- 2.1. Virus strains and their sources
- 2.2. RNA extraction
- 2.3. Primer design for confirming the identity of PPV strains by cloning and sequencing
- 2.4. Microarray design and construction
- 2.5. cDNA synthesis of total plant RNA
- 2.6. Fluorescent labeling of cDNA with cyanine 3 or cyanine 5
- 2.7. Hybridization
- 2.8. Scanning
- 3. Results
- 3.1. Identification of PPV isolates used for hybridization following cloning and sequencing of the coat protein gene
- 3.2. Total RNA extraction and cDNA synthesis
- 3.3. Design of 70-mer oligonucleotide probes
- 3.4. Detection and genotyping of PPV strains
- 4. Discussion
- Acknowledgements
- References
