A two-end point analysis reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of maize chlorotic mottle virus (MCMV)

Maize lethal necrosis (MLN) disease is a major constraint on maize production in Eastern Africa since its first report in 2012 in Kenya. The main causative agent is maize chlorotic mottle virus (MCMV) which co-infects maize with other viruses from the family Potyviridae. In Africa, Sugarcane mosaic virus (SCMV) is the most common potyvirus that co-infects maize synergistically with MCMV. MCMV can be transmitted by insect vectors, mechanically and via contaminated seeds. Monitoring of MCMV is important in farmers’ fields, seed fields, seed-lots and in grain. Robust diagnostic tools are essential in epidemiological studies, germplasm screening and exchange of disease-free materials across the regions and globally. Therefore, a sensitive, reliable and affordable diagnostic tool for MCMV is necessary in the laboratory and also in the field. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with two-end point analysis was developed to detect MCMV in active vegetative stages and in seed. Six sets of specific primers were designed and evaluated. Amplification was detected in 60 min using the SYBR green colour change and in 10 to 20 min for real-time amplification in the Genie II platform. The assay discriminated the common viruses infecting maize in Eastern Africa. The assay showed excellent specificity to MCMV. The simplicity, rapidity, and inexpensiveness of this technique make it a suitable choice for large-scale sample processing, especially by laboratories with limited resources and for field analysis performed by regulatory agencies in the region. Key word: Maize lethal necrosis, maize chlorotic mottle virus, diagnostics, loop mediated isothermal amplification.