DETECTION AND IDENTIFICATION OF PRUNE DWARF VIRUS AND PLUM POX VIRUS BY STANDARD AND MULTIPLEX RT-PCR PROBE CAPTURE HYBRIDIZATION (RT-PCR-ELISA)

S.A. Youssef, A.A. Shalaby, H.M. Mazyad, A. Hadidi

Abstract


Prune dwarf virus (PDV) and Plum pox virus (PPV) are two of the most common viruses infecting stone fruit trees, which are economically important worldwide. To improve the detection of PDV and PPV by PCR technology, we investigated the possibility of detection of these viruses in standard and multiplex RTPCR- ELISA assays. Total RNA was extracted from cherry, peach, or plum leaves and cherry pollen of PDV-infected or uninfected trees. Total RNA was also extracted from PPV-infected or uninfected leaves. Samples were extracted using commercially available RNA extraction kits. A RT-PCR-ELISA assay was developed for the detection of PDV in a single reaction or with PPV in a multiplex reaction. The use of a PDV-specific capture probe or a PPV-specific capture probe allowed the detection of each virus in a single RT-PCR-ELISA assay. Simultaneous use of the PDV-specific and PPVspecific capture probes permitted the sensitive detection of both viruses in a multiplex RT-PCR-ELISA assay. Nucleotide sequence analyses of the cloned RTPCR fragments of PDV or PPV obtained from total RNA extracts of infected leaves from different geographical locations revealed > 90% identity with published sequences, which confirmed the identity of each virus isolate investigated.

Keywords


PDV; PPV; RNA extraction; detection; RT-PCR-ELISA; amplification; DIG-labeling; cDNA capture probe; nucleotide sequence

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DOI: http://dx.doi.org/10.4454/jpp.v84i2.1094

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