Abstract:[Background] Cymbidium ringspot virus (CymRSV) is an important phytosanitary virus, which is seriously harmful in the world. [Objective] To establish a specific and sensitive assay for the quantitative analysis of CymRSV. [Methods] According to the highly conserved region in five coat protein gene sequences of CymRSV, we designed three pairs of primers and three probes, and then obtained the target sequence (145 bp) and screened out the optimal primers and probe. We then used the target sequence as a template to construct a positive recombinant plasmid and established a standard curve, on the basis of which the sensitivity, specificity, stability, and performance of the established assay were explored. [Results] In this study, the established RT-qPCR detection method for CymRSV showed that the minimum limit of detection of 1 copy for the positive plasmid standards and the minimum limit of stable detection of 5 copies/μL, demonstrating the sensitivity 103–104 times that of RT-PCR. The standard curve was y=−3.332x+40.371 (R2=0.999), which showed a good linear relationship between Ct value and logarithm of copy number and the amplification efficiency was 99.6%. The assay showed good specificity as it generated no amplification curve for other 5 common viruses. The intra-group and inter-group coefficients of variation of Ct value were less than 0.6%, which suggested good repeatability and stability. Furthermore, the established assay was employed to detect 20 samples of 4 orchid species. The positive control showed an amplification curve at Ct of 23.31, and the negative control and the samples showed no amplification curves. [Conclusion] The successful establishment of CymRSV RT-qPCR detection method based on TaqMan probe can provide technical support for the accurate detection and scientific prevention and control of orchid CymRSV virus.
