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Development of a generic PCR detection method for tobraviruses

Jones, D., Farreyrol, K., Clover, G.R.G., Pearson, M.N.
Australasian plant pathology 2008 v.37 no.2 pp. 132-136
DNA primers, DNA-directed RNA polymerase, Pepper ringspot virus, Tobacco rattle virus, annealing, complementary DNA, genes, magnesium chloride, plant tissues, plasmids, polymerase chain reaction, reverse transcription, temperature, viruses
A generic PCR assay was developed to detect all known members of the genus Tobravirus, using two forward and three reverse PCR primers with little or no degeneracy. These primers amplified a fragment of the 194K RNA polymerase gene in the RNA-1 section of the bipartite tobravirus genome from plant tissue infected with Tobacco rattle virus, Pea-early browning virus and Pepper ringspot virus. All six primer combinations produced a band of the expected size when used with cDNA derived from plant tissue infected with each of the three viruses, following reverse-transcription using either a poly-T or specific primer. A single primer pair was selected for further optimisation of annealing temperature and MgCl₂ concentration, and the sensitivity was assessed using serial dilutions of either virus-infected tissue or a purified plasmid containing the 194K RNA polymerase gene from TRV. This primer pair was able to detect all three tobravirus species using a single PCR protocol.