Oligonucleotide microarray-based detection and genotyping of Plum pox virus

• Published in: Journal of virological methods Vol. 147; no. 1; pp. 118 - 126
• Main Authors: Pasquini, Graziella, Barba, Marina, Hadidi, Ahmed, Faggioli, Francesco, Negri, Rodolfo, Sobol, Iris, Tiberini, Antonio, Caglayan, Kadriye, Mazyad, Hamed, Anfoka, Ghandi, Ghanim, Murad, Zeidan, Mohammad, Czosnek, Henryk
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 2008; Amsterdam Elsevier; New York, NY
• Subjects: 70-mer oligonucleotide probe; Biological and medical sciences; Diagnosis; DNA Probes; Fundamental and applied biological sciences. Psychology; Genotype; Genotyping; Microarrays; Microbiology; Nicotiana benthamiana; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis - methods; Plant Diseases - virology; Plant Leaves - virology; Plum pox virus; Plum Pox Virus - classification; Plum Pox Virus - genetics; Plum Pox Virus - isolation & purification; PPV strains; PPV-C; PPV-D; PPV-EA; PPV-M; Prunus; Techniques used in virology; Virology; PPV-C; PPV-D; PPV-EA; Genotyping; 70-mer oligonucleotide probe; Plum pox virus; Microarrays; PPV strains; PPV-M; Diagnosis; Typing; Plant pathogen; Microbiology; Genotype; Method; Strain; Virology; Virus; Potyvirus; Detection; Potyviridae
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2007.08.019

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.