Simultaneous detection of cymbidium mosaic virus and odontoglossum ringspot virus by TaqMan duplex real-time fluorescence quantitative PCR
To establish an efficient method for synchronous and quantitative detection of cymbidium mosaic virus(CymMV)and odontoglossum ringspot virus(ORSV),primers and probes were designed and screened according to the highly conserved regions of CymMV and ORSV CP genes.156 bp and 148 bp target sequences and the corresponding optimal specific primers and probe were obtained,and the TaqMan duplex real-time fluorescent quantitative PCR were established.The minimum detection limit was 1 copy or 6.2×10-3 fg.The minimum stable detection limit was 10 copies or 6.2×10-2 fg,whose sensitivity is 10-100 times as much as that of RT-PCR.The standard curve showed a good linear relationship with the amplification efficiencies of 97.7％and 100.2％,respectively,and the correlation coefficient R2 of 1.000 and 0.999,respectively.There was no amplification curve for the other five common viruses,indicating the method was very specific.The intra-and interassay CVs of Ct values was less than 0.60％,indicating the method was of good repeatability.In addition,66 leaves from four different orchid genera were analyzed using the established method and gene chip assay,and the detection rate of this method was 53.85％(CymMV)and 162.5％(ORSV)higher than that of gene chip assay,respectively.In conclusion,the TaqMan duplex assay established in this study can detect CymMV and ORSV simultaneously with reliable results,which has a board prospect of application and can provide technical support for accurate virus identification,scientific prevention and control,and containment of virus transmission from the source.
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