Multiplex RT-PCR detection of three common viruses infecting orchids

A multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection of three orchid viruses: cymbidium mosaic virus (CymMV), odontoglossum ringspot virus (ORSV), and orchid fleck virus (OFV). Primers were used to amplify nucleocapsid protein gene fragments of 845 bp (ORSV), 505 bp (CymMV) and 160 bp (OFV). A 60-bp amplicon of plant glyceraldehyde-3-phophate dehydrogenase mRNA was included as an internal control against false negatives. The assay was validated against 31 collected plants from six orchid genera and compared with results obtained by transmission electron microscopy (TEM). The RT-PCR assay proved more sensitive than TEM for detection of OFV.