Simultaneous detection of stone or pome fruit viroids by single tube-RT-PCR

Stone fruit viroids such as peach latent mosaic (PLMVd) and hop stunt (HSVd) as well as pome fruit viroids that include apple scar skin (ASSVd), pear blister canker (PBCVd) and apple dimple fruit (ADFVd) are widely distributed and economically important. In order to obtain a rapid and efficient detection method of these viroids, we have developed a single tube RT-PCR protocol that amplifies simultaneously ASSVd, PBCVd and ADFVd or PLMVd and HSVd from their respective hosts. Multiple alignment and comparison of the nucleotide sequences of ASSVd, PBCVd and ADFVd allowed us to select a conserved region among these viroids and design a primer pair for their simultaneous amplification in a single PCR reaction. These new designed primers amplify a specific DNA fragment which contains a major portion of each viroid: ASSVd 267 bp, PBCVd 261 bp and ADFVd 252 bp. We were not able, however, to select and design common primers for the detection of both PLMVd and HSVd. For this reason we used a mixture of two primer pairs, each pair for one pathogen, to amplify PLMVd and HSVd in a single PCR reaction. The development of this RT-PCR protocol enabled us to identify in a single PCR reaction the most widely distributed and economically important viroids that affect pome or stone fruit germplasm. This assay should facilitate the control of these temperate fruit tree viroids as it is rapid and inexpensive.