ISHS Acta Horticulturae 386: XVI International Symposium on Fruit Tree Virus diseases
DETECTION OF PLUM POX POTYVIRUS (PPV) BY DNA ENZYME IMMUNO-ASSAY |
| Authors: | M. Schönfelder, G. Adam, E. Maiss |
| Abstract:
The DNA enzyme immunoassay (DEIA, Trademark of Sorin Biomedica, Italy) was used to detect PPV-specific dsDNA pieces amplified by polymerase chain reaction (PCR) after immunocapturing virions with polyclonal rabbit IgG from PPV-infected tobacco and plum. For PCR-amplification, two 18-mer primers located within the coat protein region were used which led to the synthesis of a 806 bp fragment. The PCR-product was detected by a 44-mer oligonucleotide complementary to viral RNA that resided inside the primer-defined boundaries. This capture oligonucleotide was 5'-biotinylated and could thus be linked to a microtiter plate coated with streptavidin. Hybrid molecules formed between the capture oligonucleotide and the PCR-products were finally detected by an dsDNA-specific monoclonal antibody and subsequent incubation with an anti-mouse rabbit IgG labeled with alkaline phosphatase. The detection of the PCR-products by the dsDNA-specific antibody was six-times more sensitive than the detection by agarose gel electrophoresis. When the DEIA procedure was compared with DAS-ELISA, it revealed a higher sensitivity and exceeded the dilution endpoint of ELISA by 2 two-fold dilutions. This resulted in a higher test-fidelity especially when mixed samples were assayed. | |
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