Multiplex RT-PCR method for the simultaneous detection of nine grapevine viruses.

Viral diseases are a serious pathological problem for grapevines, and in recent years the need for increasingly specific and rapid diagnostic methods for the selection of propagation materials has grown. Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Grapevine rupestris stem pitting-associated virus, Grapevine fleck virus, and Grapevine leafroll-associated viruses 1, 2, and 3 are nine of the most widespread viruses that naturally infect grapevines. A multiplex RT-PCR was developed for simultaneous detection of these nine grapevine viruses, in combination with a plant RNA internal control used as an indicator of the effectiveness of the reaction. One to ten fragments specific for the viruses and an internal control were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel. The protocol reported is an update of previously published protocols for RNA extraction and multiplex diagnosis of viruses. After several years of use and hundreds of samples tested, and following validation in several laboratories, this multiplex RT-PCR provides a reliable and rapid method for detecting grapevine viruses from a large number of samples.