Detection and identification of sweet potato viruses by the polymerase chain reaction

Among the numerous agents infecting tropical crops, such as sweet potato, several viruses belonging to the potyvirus group have been described Two degenerate oligonucleotide primers, derived from conserved regions of the genome of potyviruses, have been designed in order to amplify a fragment including the nonconserved 5' terminal part of the coat protein cistron (which may greatly differ in length in distinct potyviruses) and the more conserved 3' terminal part of the RNA polymerase cistron. A combined assay of reverse transcription followed by the Polymerase Chain Reaction using these primers on total RNA extracted from different sweet potato clones from China, yielded one or several of three fragments, thus suggesting the presence of at least one to three distinct potyvirus, according to the sweetpotato clone. Sequence analysis of the three fragments confirmed the presence of mixed infections by distinct potyviruses in the sweet potato clones investigated. The cloned PCR products were used to develop a non-radioactive hybrization test, using the digoxigenin labelling system. The PCR procedure using degenerate primers is thus a rapid and valuable tool for the detection and characterization of potyviruses. The amplified fragments thus obtained can easily be cloned into a vector and sequenced for classification and taxonomical studies, or used for the development of nucleic acid hybridization tests to be used in certification procedure.