Improved PCR procedures for multiple identification of some artichoke and grapevine viruses.

Reverse transcription polymerase chain reaction (RT-PCR) amplification was adapted for detection of either single or mixed viral infections in grapevine and artichoke. DNA primers specific for grapevine A closterovirus (GVA), grapevine B trichovirus (GVB), or grapevine leafroll-associated closterovirus III (GLRaV-III) were used for RT-PCR amplification of specific genomic segments from GVA, GVB and GLRaV-III-infected grapevine tissues. The reactions produced discrete fragments of 0.43, 0.45 and 0.34 kb which were representative of GVA, GVB and GLRaV-III, respectively. Immunocapture RT-PCR (IC-RT-PCR) or multiplex RT-PCR (M-RT-PCR) were utilized for the detection of these viruses in extracts of infected grapevine leaves, dormant cuttings and/or viruliferous mealybugs. IC-RT-PCR was more efficient than RT-PCR for the detection of GVA in viruliferous mealybugs, whereas M-RT-PCR was easier and faster than IC-RT-PCR for the detection of GLRaV-III and GVB in infected grapevine tissues. Six specific primers were used for RT-PCR amplification of specific genomic segments of artichoke mottled crinkle tombusvirus (AMCV), artichoke Italian latent nepovirus (AILV) and artichoke latent potyvirus (ALV) in artichoke plant sap. This set of primers was also successfully used for the simultaneous detection of the three viruses in cases of mixed infections, because each primer pair retained its specificity and reverse transcription-amplification reactions ran independently, yielding products of the expected virus-specific size. The limits of simultaneous detection were about 400 fg for AMCV and ALV and 4 pg for AILV, whereas the limits of detection in separate reactions were about 100fg for all three viruses.