The multiplex RT-PCR assay developed in this study providesa sensitive, simple, rapid, and cost-effective method to detectsimultaneously the four TBTD complex agents in one reaction. Theassay was proved to be reliable. It will be useful to detect large-scale samples during the disease survey in the fields. Furthermore,application of this method could facilitate future research on theetiology of TBTD, disease outbreak monitoring and interactionsamong viruses, hosts and vectors.