Oligonucleotide microarray-based detection and genotyping of Plum pox virus.
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.
Pasquini
Graziella
G
CRA-Istituto Sperimentale per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Rome, Italy.
Barba
Marina
M
Hadidi
Ahmed
A
Faggioli
Francesco
F
Negri
Rodolfo
R
Sobol
Iris
I
Tiberini
Antonio
A
Caglayan
Kadriye
K
Mazyad
Hamed
H
Anfoka
Ghandi
G
Ghanim
Murad
M
Zeidan
Mohammad
M
Czosnek
Henryk
H
D015342:DNA Probes; D005838:Genotype; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010935:Plant Diseases / Q000821:virology*; D018515:Plant Leaves / Q000821:virology; D017801:Plum Pox Virus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*
D015342:DNA Probes