Detection of Five Seedborne Legume Viruses in One Sensitive Multiplex Polymerase Chain Reaction Test

A reverse transcription polymerase chain reaction (RT-PCR) assay has been developed that can simultaneously test a sample, in one tube, for the presence of five seedborne legume viruses that are of great concern to legume germ plasm banks: alfalfa mosaic alfamovirus, bean yellow mosaic potyvirus, clover yellow vein potyvirus, cucumber mosaic cucumovirus, and subterranean clover mottle sobemovirus. RT-PCR assays were also developed for the detection of subterranean clover redleaf luteovirus and subterranean clover stunt virus, thus providing a detection system for all well-characterized viruses known to infect subterranean clover. Primers were designed so that the size of the RT-PCR product was indicative of the virus amplified and, where sequences of more than one strain of virus were available, conserved regions were given preference as primer targets