In this study, a generic ramped-annealing (RAN) nested RT-PCR was developed, allowing the simultaneous detection and fast characterization of ilarviruses. The method involves a one-step RT-PCR in which a pair of degenerate primers amplifies a 381-bp part of the polymerase gene (RNA2), followed by a nested PCR amplification that increases detection sensitivity. The sensitivity and detection range of the method were further increased by applying a ramped annealing thermocycling step both in the first RT-PCR and in the subsequent nested PCR. The 371-bp nested amplicons can be sequenced directly, without cloning, to obtain initial sequence information on ilarvirus genomes, or can undergo a restriction enzyme analysis for rapid identification of already known virus species. Phylogenetic relationships among different members of the family Bromoviridae were inferred with maximum likelihood and Bayesian analysis, using published homologous partial amino acid sequences corresponding to the nested amplicon and also to a longer residue data set (432–453 aa) comprising all possible positions of homology among the RNA2-encoded polymerases of members of the family Bromoviridae . The implications of these analyses on the taxonomy of ilarviruses are discussed. The specific partial polymerase sequence, corresponding to the polymerase core palm structure (motifs A–D), was verified as phylogenetically informative and can be used to separate ilarviruses from other members of the family Bromoviridae , providing initial information for ilarvirus species characterization. However, the phylogenetic signal of this region is not reliable for inferring relationships among distantly related ilarviruses.
Archives of Virology – Springer Journals
Published: Sep 1, 2007
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