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Narcissus (Narcissus spp.) is an important commercial crop and millions of narcissus bulbs are traded across the world annually. Three major viruses, namely, Narcissus yellow stripe virus (NYSV), Narcissus latent virus (NLV) and Narcissus mosaic virus (NMV), have substantial impacts on global crop yields of narcissus. A multiplex RT-PCR assay was developed and used to simultaneously detect these three viruses. Three pairs of virus-specific primers were designed according to the conserved regions of the coat protein (CP) genes, generating expected fragments of 668 bp, 554 bp and 372 bp for NYSV, NLV and NMV, respectively. Specificity and compatibility of primer pairs were tested using uniplex and multiplex RT-PCR. Primer concentration, annealing temperature and cycle number were optimized to enable efficient and balanced amplification of the three viruses. The optimized multiplex RT-PCR was as sensitive as uniplex RT-PCR. Field samples were collected to validate the method, and all the viruses were successfully detected, thereby confirming the effectiveness and reliability of the developed multiplex RT-PCR. This method could be used for large-scale surveys of these three viruses in narcissus and thus might play a role in diagnosis and prevention of these pathogens. [ABSTRACT FROM AUTHOR]