Simultaneous multiplex RT-PCR detection of four viruses associated with maize lethal necrosis disease

Maize lethal necrosis disease (MLND) is a serious disease of worldwide importance. It is caused by the co-infection of maize with maize chlorotic mottle virus (MCMV) and a potyvirus, such as sugarcane mosaic virus (SCMV), that acts synergistically to produce more severe symptoms and production losses. More recently, maize yellow mosaic virus (MaYMV) and maize-associated totivirus (MATV) were found to co-infect with MCMV and SCMV in maize plants. To facilitate the detection of these viruses in co-infected maize, a multiplex RT-PCR assay was developed in this study. The assay used five specific primer pairs and simultaneously amplified these four viruses as well as the elongation factor 1α (EF 1α) gene use as internal control in one tube. The concentration of the primers, annealing temperature, annealing time, extension time and amplification cycles were optimized for the multiplex RT-PCR. The detection limit of the assay was up to 100 pg of total cDNA template. This multiplex RT-PCR assay was shown to be a sensitive and effective tool for the screening of field samples for the presence of these viruses in co-infected maize.