Multiplex Reverse Transcriptase-Polymerase Chain Reaction Applied To Virus Detection In Globe Artichoke

Six specific primers were used for reverse transcription-amplification of parts of the genomic RNAs of artichoke mottled crinkle tombusvirus (AMCV), artichoke Italian latent nepovirus (AILV) and artichoke latent potyvirus (ALV) in artichoke plant sap. Each primer pair was highly specific for each viral RNA template and in multiplex reverse transcription-amplification reactions run independently, yielding products of the expected virus-specific size. The optimal artichoke plant sap dilution was about 103-fold in 10 mM DTT and the optimal MgCl2 concentration was 2.25 mM. The detection limits for simultaneous amplification were of about 400 fg for AMCV and ALV and of about 4 pg for AILV. The method proved to be useful for simultaneous detection in cases of viral mixed infections which are likely to occur in nature.