A Reliable and Rapid Multiplex RT-PCR Assay for Detection of Two Potyviruses and a Pararetrovirus Infecting Canna Plants.

ABSTRACT

Canna plants are subject to serious virus diseases. The three most common viruses identified in canna plants are Bean yellow mosaic virus, Canna yellow mottle virus, and Canna yellow streak virus. Recent studies indicate that canna plants are commonly infected with more than one virus. Thus, the efficient control of these viruses in canna plants requires the availability of a reliable method for detecting mixed virus infection. This report presents a two-step multiplex reverse-transcription polymerase chain reaction (RT-PCR) that was developed to simultaneously detect two potyviruses and one pararetrovirus genome. We optimized the method for nucleic acid isolation for managing a large population of samples, and the primer concentrations to ensure sensitivity and reliability of the assay, and determined the detection limit in simplex and multiplex RT-PCR assays using plasmid controls and nucleic acids isolated from virus-infected plants. Combined with an automated method for total nucleic acid isolation, this multiplex RT-PCR procedure could be routinely used for virus detection in research and diagnostic laboratories.