ISHS Acta Horticulturae 385: VII International Symposium on Small Fruit Virus Diseases
IMMUNOCAPTURE POLYMERASE CHAIN REACTION ASSAY AND ELISA FOR THE DETECTION OF STRAWBERRY MILD YELLOW EDGE ASSOCIATED POTEXVIRUS |
| Authors: | D. KADEN-KREUZIGER, S. LAMPRECHT, R.R. MARTIN, W. JELKMANN |
| Abstract:
Five virus isolates were investigated: MY-18 from Rubus rosifolius and four strawberry isolates originating from North America, United Kingdom, and Germany. Using primer combinations at the viral 3'-terminal half, a band of either 406 bp or 883 bp was amplified. Several techniques were investigated for the extraction of nucleic acid from R. rosifolius leaf tissue; the method according to Doyle & Doyle (1990) and mRNA isolation with Dynabeads oligo(dT)25 was successful for RNA template preparation. Virus detection from strawberry tissue was only accomplished using an immunocapture reverse transcriptase PCR (IC-RT-PCR). Antisera prepared from a fusion of the viral coat protein to either protein A or glutathione S-transferase that had been successfully used in ISEM failed in IC-RT-PCR and ELISA. Effective RNA template preparation from strawberry and R. rosifolius tissue was achieved using an antiserum to a recombinant protein with only six N-terminal histidine residues. Using this antiserum in ELISA, SMYEAV was successfully detected from strawberry tissue. | |
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