Detection and Molecular Characterization of Grapevine Virus A in Jordan

In a study on grapevines in Jordan conducted between 2002 and 2003, grapevine virus A (GVA) was detected in all areas where grapevines were planted. DAS-ELISA analysis of samples from symptomatic trees found that 16.1% of samples were infected with GVA. Using a GVA- specific primer pair (H587/C995), a portion of the coat protein gene of the virus was amplified by IC-RT-PCR and RT-PCR, using leaf extracts and RNA extracted from infected grapevines respectively. After cloning and sequencing the coat protein gene of the Jordanian isolate of GVA (GVA-Jo), the sequence of the amplified product was compared with sequences of other GVA isolates from different countries

Occurrence and Distribution of Citrus tristeza virus (CTV) in the Jordan Valley

In a survey conducted in 2002 and 2003, Citrus tristeza virus (CTV) was detected in the Jordan Valley. The direct tissue blot immunoassay (DTBIA) indicated that 12.7 and 15.2% of samples tested in the central and northern Jordan Valley respectively were infected with CTV. Similar results showed that all citrus species grown in the Jordan Valley were susceptible to CTV. DAS-ELISA analysis of samples from a citrus orchard in the Dir Alla area with severe CTV symptoms indicated that 49% of samples were infected with CTV. Using a CTV specific primer pair (CTV1/CTV10), the coat protein gene of the virus was successfully amplified from leaf extracts obtained from CTVinfected trees by IC-RT-PCR. After cloning and sequencing the coat protein gene, the sequence of the amplified product was deposited in the GenBank

Molecular characterization of Grapevine virus A isolates from Jordan

A total of 1141 samples of petioles and canes of grapevines from different locations in Jordan were tested by DAS-ELISA for Grapevine virus A (GVA). About 14.2% of samples were infected with GVA. Using Reverse Transcription- Polymerase Chain Reaction (RT-PCR) or Immunocapture (IC)-RT-PCR, a fragment of 430 bp of the coat protein gene was amplified using the primer pair H587/C995. Fifteen clones were sequenced and aligned with each other and with the Jordanian isolate (GVA-Jo) using the DNAMAN program. Alignment analysis showed that all Jordanian isolates shared 100% nucleotide identity with each other and with the Jordanian isolate GVA-Jo. To classify the collected GVA isolates in one of the GVA groups a pairwise nucleotide alignment between isolate GVA-5R and isolates from South Africa; 92/778, JP98 and P163-1 representing GVA group I, II and III respectively was done. Alignment results indicated that isolate GVA-5R shared 90, 83, and 76% nucleotide similarity with GVA groups I, II, and III, respectively

Validation of a microarrays protocol for detection and genotyping isolates of Plum pox virus

A genomic strategy for PPV identification has been recently developed (Pasquini et al., 2008). The method is based on using a 70-mer oligonucleotide DNA microarray chip capable of simultaneously detecting and genotyping PPV strains. Universal and specific probes have been identified and used with a sensitive protocol of hybridization using an indirect fluorescent labelling of cDNA product with cyanine able to enhance the sensitivity of the virus detection avoiding the use of the PCR amplification step. In order to evaluate the protocol fitness for diagnostic use, about 30 samples belonging to a PPV isolates collection, including M, D, EA and C strains, have been used for its validation, that was determined, estimating the performance criteria that include the following parameters: diagnostic sensitivity (D-SN), diagnostic specificity (D-SP) and diagnostic accuracy (D-AC). Keywords: oligonucleotides chip, PPV, sensitivity, specificity, accuracy, performance criteri

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