Abstract: Using the‘Red Globe’infected by four grapevine viruses as a sample，the concentrations of dNTPs，Taq DNA polymerase，the primers，annealing temperature and template quantity were optimized for the simultaneous detection of four viruses Grapevine leafroll-associated virus-3（GLRaV-3），Grapevine rupestris stem pitting associated virus（GRSPaV），Grapevine virus B（GVB），Grapevine virus A（GVA） by multiplex RT-PCR. Comparison of the sensitivity of both multiplex-and single RT-PCR for the detection of those viruses showed no significant differences. The target fragments with expected sizes of 905 bp（GRSPaV），546 bp（GLRaV-3），460 bp（GVB）and 196 bp（GVA）were amplified from the sample. The multiplex RT-PCR products of those viruses were cloned and sequenced. Result showed that the obtained sequences had high similarities to reported corresponding sequences of those viruses. The viral detection results of seven grapevine samples in field suggested that the multiplex RT-PCR could be used for the simultaneous detection of those four viruses.

Key words: multiplex RT-PCR, detection, Grapevine leafroll-associated virus-3, Grapevine rupestris stem pitting associated virus, Grapevine virus B, Grapevine virus A