DETECTION OF POME FRUIT VIROIDS BY RTPCR USING A SINGLE PRIMER PAIR
F. Faggioli, E. Ragozzino
Apple scar skin viroid (ASSVd), Pear blister canker viroid (PBCVd), and Apple dimple fruit viroid (ADFVd) are widely distributed and economically important pome fruit viroids, all belonging to genus Apscaviroid. For a rapid and efficient detection method of these viroids, we have developed a one-tube RT-PCR protocol that amplifies simultaneously ASSVd, PBCVd and ADFVd from their respective hosts. Multiple alignment and comparison of the nucleotide sequences of ASSVd, PBCVd and ADFVd allowed us to select a conserved region and design a primer pair for their polyvalent amplification in a single RT-PCR reaction. These newly 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. The development of this RT-PCR protocol enabled us to identify different isolates of the three viroids and to evaluate their distribution in cultivated and wild pome fruit germplasm.