Application of the immunocapture-PCR technique for plum pox potyvirus detection under field conditions in Greece and assays to simplify standard techniques

摘要

The immunocapture-polymerase chain reaction (IC-PCR) technique was used for plum pox potyvirus (PPV) detection. The expected gain in sensitivity of at least 2000-fold over ELISA, was confirmed with greenhouse samples. The effectiveness under field conditions in Greece was assayed by a direct comparison with ELISA on 70 symptomless leaves of an infected apricot tree and on 56 apricot mother trees with 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. RFLP analysis of the PCR products revealed that all but 1 isolate belonged to the M-type PPV. Simplification of the standard technique by applying print-capture PCR was quite effective with greenhouse samples but less effective with field samples. Microplate hybridization using a DIG-labelled probe for amplicon revelation was 100-fold more sensitive than agarose electrophoresis.