Construction and Use of a Cloned cDNA Probe for the Detection of Plum Pox Virus in Plants

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Abstract
Complementary DNAs to plum pox virus (PPV) were cloned into Escherichia coli plasmid pUC9. Clones containing about 1 kb of the 3'' region of PPV RNA were obtained. Subcloning of a 720-bp fragment lacking the 3'' poly-A region into pBR322 provided a recombinant plasmid named pPPV9A. This plasmid was labeled by nick translation and used in dot-blot hybridization assays. As little as 100 pg of purified virus was detected. This is equivalent to about 5 pg of PPV RNA. The enzyme-linked immunosorbent assay (ELISA) applied to the same purified virus solution allowed the detection of about 1 ng of virus per assay and was therefore 10 times less sensitive. When the respective sensitivities of the two methods were compared with crude sap [pea, peach] samples, molecular hybridization was again more sensitive as far as actual amounts of virus or of infected tissues were concerned. The usefulness of this technique for routine detection work is discussed.