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This work aims to characterize and detect the apple stem grooving virus (ASGV). Amplification products obtained by RT-PCR using degenerate primers targeting a conserved region of the 3'end of the RNA polymerase gene have been sequenced. The knowledge of the partial genomic sequences of several European isolates allowed us to search for conserved regions in order to design specific primers to be used in RT-PCR experiments. For these specific primers, the different steps of the RT-PCR amplification and the methods of preparation of the extracts containing the target viral sequences have been optimized to achieve the reproducible detection of the viral infection from leaves or stems of woody plants. The best results have been obtained by using a one step RT-PCR protocol combining the reactions of retrotranscription of the viral RNA with AMV reverse transcriptase and DNA chain polymerization with a mixture of Taq and PWO DNA polymerases. That refined technique is applied to total RNA, ds RNA or diluted crude extracts. Different techniques of detection of the amplification products were compared for their sensitivity and specificity. The best results were obtained by trapping the complex made between the amplification product and an internal digoxigenin-labeled detection probe on a microtitration plate with a biotine labeled probe. This method allows the detection of the virus all over the year, and especially during the summer and from dormant wood when the reference serological test is not efficient.

Malus --- Malus --- Plant viruses --- Plant viruses --- diagnosis --- diagnosis --- Molecular genetics --- Molecular genetics --- DNA. --- DNA --- RNA. --- RNA --- genetic engineering --- genetic engineering