The diagnosis of the tomato variant of pepino mosaic virus:: An IC-RT-PCR approach

An immunocapture-retrotranscription-PCR (IC-RT-PCR) procedure for the detection of the tomato-infecting variant of pepino mosaic potexvirus (PepMV) was developed, following an approach that did not require knowledge of viral sequence, availability of commercial antibodies to the virus, or purification of viral particles. Degenerate PCR primers, whose design was based on alignments of published potexviruses sequences to prime theoretically the amplification of a viral genomic fragment of any potexvirus, were used to synthesize cDNA of the potato virus X (PVX) and PepMV RNA polymerases. Tubes coated with antibodies against double-stranded RNA were used in the initial amplifications. Two different non-overlapping fragments of the PepMV polymerase gene were cloned and sequenced, and their putative positions in the viral RNA were determined relative to the PVX sequence. For the diagnosis procedure, new specific PepMV primers were designed based on the virus sequence obtained and their utility to amplify a unique diagnostic band of 835 bp after IC-RT-PCR with specific anti-PepMV antibodies was shown. The method developed should allow the rapid diagnosis of a virus that seriously threatens tomato cultivation in several European countries.