Abstract
A real-time multiplex RT-PCR has been developed for the simultaneous detection and identification of the
major RNA viruses that infect grapevines (Grapevine fanleaf virus, Arabis mosaic virus, Grapevine leafrollassociated
virus 1, Grapevine leafroll-associated virus 3 and Grapevine fleck virus). Serial dilutions of infected
plant extracts were tested using the new method, and the results were compared with those obtained
using a commercially available ELISA and real-time singleplex RT-PCR. The two real-time RT-PCR versions
detected up to the same level of dilution and were at least 10,000 times more sensitive than the ELISA.
In addition, 158 grapevine plants collected in a survey of the Protected Designation of Origin in Alicante,
Spain were compared using the three methods. The results of the molecular methods were very similar,
with only four discordant results, and both were able to detect many more infected plants than the
ELISA. The high prevalence of Grapevine fleck virus, Grapevine leafroll-associated virus 3 and Grapevine
fanleaf virus suggests that the main pathways of viral introduction are infected plant material that has
escaped controls and/or uncontrolled traffic of propagating plant material. Real-time multiplex RT-PCR
could be used to facilitate a better control of grapevine viruses
