Narcissus yellow stripe virus and Narcissus mosaic virus detection in Narcissus via multiplex TaqMan-based reverse transcription-PCR assay

Abstract

Aims: Development of a multiplex TaqMan RT-qPCR assay to simultaneously detect Narcissus yellow stripe virus (NYSV) and Narcissus mosaic virus (NMV), frequently causing mixed narcissus infection. Feasibility verification was confirmed in natural samples.

Methods and results: Primers and probes were designed based on the conserved CP gene regions of NYSV or NMV and their suitability for singleplex and multiplex TaqMan RT-qPCR assays as well as for conventional RT-PCR. Conventional RT-PCR, singleplex and multiplex TaqMan RT-qPCR assays proved to be NYSV and NMV specific. P-values and coefficients of variation of TaqMan RT-qPCR assays indicated high reproducibility. Significantly increased sensitivity was achieved compared to conventional RT-PCR. The detection limit of both viruses was 10³ copies with superior correlation coefficients and linear standard curve responses between plasmid concentrations and Ct values. NYSV and NMV infection of narcissus leaves, petals and bulbs could successfully be detected via our multiplex RT-qPCR method at 1·25 mg.

Conclusion: Our multiplex TaqMan RT-qPCR assay provides rapid, specific, sensitive and reliable testing to simultaneously detect NYSV and NMV, supplying useful routine monitoring for different narcissus samples.

Significance and impact of the study: Efficient identification and discrimination of the narcissus viruses provides reliable information for scientists and conventional growers. Furthermore, it enriches the information of NYSV, NMV and other narcissus viruses.

Keywords: Narcissus mosaic virus; Narcissus yellow stripe virus; detection limit; mixed infection; multiplex probe-based method; narcissus viruses; simultaneous detection.