|Riassunto in Inglese:|
A genomic strategy for Plum pox virus (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 (Pasquini et al, 2008).
In order to evaluate the protocol fitness for diagnostic use, 30 samples belonging to a PPV isolates collection, including M, D, EA and C strains, have been used for its validation, that was determined by estimating performance criteria that include the following parameters: diagnostic sensitivity (D-SN), diagnostic specificity (D-SP) and diagnostic accuracy (D-AC) (Pasquini et al., 2009).