@ARTICLE{TreeBASE2Ref2110, author = {Filomena Fonseca and Gustavo Nolasco and Carla Santos and Gon?alo Silva}, title = {Development of an asymmetric PCR ELISA typing assay for citrus tristeza virus based on the coat protein gene.}, year = {2008}, keywords = {}, doi = {}, url = {}, pmid = {}, journal = {Journal of Virological Methods}, volume = {}, number = {}, pages = {}, abstract = {The coat protein gene of isolates of citrus tristeza virus (CTV) from twenty citrus-producing regions around the world was amplified by RT-PCR, TA cloned, and characterized by SSCP. Haplotypes that produced different patterns within each geographic region were sequenced and a database of 153 accessions of CTV was assembled. Phylogenetic analysis revealed the existence of seven well-defined clusters (Coefficient of differentiation 0.78). An asymmetric PCR ELISA typing (APET) assay was developed in the frame of this clustering pattern using a set of eight hybridization probes. The membership of any unknown haplotype is determined by comparing its pattern of reaction against the whole set of probes and not, as previously done in hybridization assays, in an all-or-nothing basis. Interpretation of the results is objective and done through a visual basic application that compares the rates of hydrolysis of the ELISA substrate of an assayed isolate to a matrix of rates of hydrolysis obtained from standard haplotypes. This assay was validated and showed a better ability to resolve haplotypes than other assays to which it was experimentally compared. It may be automated to the same extent as any ELISA assay.} }

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