ISHS Acta Horticulturae 472: XVII International Symposium Virus and Virus-Like Diseases of Temperate Fruit Crops
APPLICATION OF THE IMMUNOCAPTURE-PCR TECHNIQUE FOR PLUM POX POTYVIRUS DETECTION UNDER FIELD CONDITIONS IN GREECE AND ASSAYS TO SIMPLIFY STANDARD TECHNIQUES |
| Authors: | C. Varveri, K. Boutsika |
| Keywords: | polymerase chain reaction, ELISA, print-capture, microplate hybridization |
| Abstract:
The immunocapture-polymerase chain reaction (IC-PCR) technique was applied for plum pox potyvirus (PPV) detection. The expected gain in sensitivity of at least 2000 fold over ELISA was confirmed with greenhouse samples. Its effectiveness under local field conditions was assayed by a direct comparison with ELISA on 70 symptomless leaves of an infected apricot tree and on 56 apricot mother trees giving ELISA readings around the detection threshold. IC-PCR detected PPV in 31% of ELISA negative leaf samples and in 23% of ELISA doubtful trees. The polyvalence of the assay was checked with 25 Greek PPV isolates which were all amplified. The RFLP analysis of their PCR products revealed that with the exception of one case they belonged to the M-type isolates of PPV. Simplification of the standard technique by applying print-capture PCR was quite effective with greenhouse samples but less with field ones. Microplate hybridization using a DIG-labelled probe for amplicon revelation was 100-fold more sensitive than agarose electrophoresis, thus indicating a great potential to be used routinely. | |
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