Development of a generic PCR detection method for tobraviruses

Abstract

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.