Multiplex RT-PCR for Simultaneous Detection of Three Viruses in Oncidium

By using specific primers designed on the basis of conserved sequences of coat protein gene,a multiplex PCR protocol for the detection of 3commonly found disease-causing viruses,i.e.,Cymbidium mosaic(CyMV),Odontoglossum ringspot(ORSV)and bean yellow mosaic(BYMV),in Oncidium was developed.Three specific fragments were simultaneously amplified in a single PCR reaction system.Their molecular weights were determined to be 571 bp for CyMV,325 bp for ORSV,and 212 bp for BYMV.Sequence analysis indicated that the 3viruses were at least 98% homogenous with their respective reference viruses.The sensitivity test on this newly developed methodology showed that it was capable of accurately detecting the 3viruses simultaneously in a bulbous iris tissue at the level of 10-2 mg.