Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.1/6208
Título: Development of an asymmetric PCR-ELISA typing method for citrus tristeza virus based on the coat protein gene
Autor: Nolasco, Gustavo
Santos, C.
Silva, G.
Fonseca, Filomena
Data: 2009
Editora: Elsevier
Resumo: The coat protein gene of isolates of citrus tristeza virus (CTV) from 20 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 hybridisation 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 hybridisation 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 compared experimentally. It may be automated to the same extent as any ELISA. (c) 2008 Elsevier B.V. All rights reserved.
Peer review: yes
URI: http://hdl.handle.net/10400.1/6208
DOI: https://dx.doi.org/10.1016/j.jviromet.2008.09.030
ISSN: 0166-0934
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