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A ligation‐anchored PCR method for the cloning of the 3’end of a tobamovirus infecting Impatiens New Guinea hybrids *
Author(s) -
Wetzel T.,
Moerbel J.,
Krczal G.
Publication year - 2000
Publication title -
eppo bulletin
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.327
H-Index - 36
eISSN - 1365-2338
pISSN - 0250-8052
DOI - 10.1111/j.1365-2338.2000.tb00927.x
Subject(s) - biology , tobamovirus , virology , genetics , microbiology and biotechnology , tobacco mosaic virus , virus
The development of the polymerase chain reaction (PCR) and PCR‐derived methods has greatly improved the sensitivity and specificity of the detection of pathogens. We have developed a ligation‐anchored PCR technique (LA/PCR) to clone the 3’end of the viral genome of a new tobamovirus infecting Impatiens New Guinea hybrids. Purified viral RNA was ligated to an EcoRI‐linearized plasmid using T4 RNA ligase, and the ligation product subjected to a reverse transcription‐PCR (RT/PCR), with a degenerate tobamovirus primer located in the tobamovirus 3’non‐coding region and the universal reverse primer corresponding to plasmidic sequence. The resulting PCR product, which contained some plasmidic sequence and the 3’end of the viral genome, was cloned and sequenced. Sequence comparisons with the corresponding sequences of other tobamoviruses revealed very high homologies with oilseed rape mosaic tobamovirus