Resumo

HERNANDEZ-RODRIGUEZ, Lester et al. Evaluation of a non-radioactive dot-blot nucleic acid hybridization as a detection tool for citrus leaf blotch virus in Cuba. Rev. Protección Veg. [online]. 2019, vol.34, n.3, e09.  Epub 01-Dez-2019. ISSN 1010-2752.

A non-radioactive dot-blot nucleic acid hybridization method was evaluated for detecting citrus leaf blotch virus (CLBV, Flexiviridae: Citrivirus) in total RNA extracts from kumquat trees. DNA fragments partially encompassing the open reading frames encoding the polymerase (RdRp) and the movement (MP coat (CP) proteins of CLBV were used to generate digoxigenin (DIG-11-dUTP) probes by PCR. Probes were individually generated using cloned DNA fragments of the Cuban CLBV isolate as templates. In the assays, several working conditions, including the use of different membranes, hybridization and washing buffers were tested. The method detected up to 40 pg of a plasmid containing the complete CLBV genome, as well as viral genomic sequences in RNA extracts of naturally infected trees of 'Nagami' kumquat (Fortunella margarita) and 'Round' kumquat (F. japonica). Results support considering the method developed an appropriated tool to be used after its validation by the programs of sanitation, quarantine, and certification of citrus propagation materials in Cuba.

Palavras-chave : budwood; certification; Citrivirus; CLBV; diagnosis; Flexiviridae; Fortunella.

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