Establishment of Multiplex RT-PCR Detection System for Three Viruses in Freesia

The specific primers were designed according to conserved sequences of coat protein (CP) gene of Freesia mosaic virus (FreMV), Cucumber mosaic virus (CMV) and Bean yellow mosaic virus (BYMV) published in GenBank, and a multiplex PCR protocol for simultaneous detection of these three viruses in freesia was developed. Three specific fragments were simultaneously amplified in a single PCR reaction. Their lengths were determined to be 340, 628 and 212 bp, respectively. The sequence analysis indicated that three viruses shared at least 97% of homology with reference sequence. Sensitivity test showed that these three viruses could be detected out in the infected plant tissue greater than 10-2 mg.