Development of loop mediated isothermal amplification assays for the detection of sugarcane white leaf disease

Abstract The Sugarcane white leaf (SCWL) phytoplasma from group 16SrXI is the causal agent of Sugarcane white leaf disease (SWLD) resulting in severe yield losses to the sugar industry. Although microscopy, serology based methods, dot blot DNA hybridization, and PCR have been developed as diagnostic tests, these techniques are labour intensive, costly and time-consuming and can only be undertaken in well-equipped laboratories. Therefore, the objective of the present study was to develop a loop-mediated isothermal amplification (LAMP) assay for SCWL as an alternative approach for quick and efficient detection of the sugarcane white leaf (SCWL) phytoplasma within 30 minutes. Three LAMP primer sets including a universal 16S rRNA based phytoplasma assay and two 16SrXI group-specific assays were used to detect the 16SrXI SCWL phytoplasma. Plant cytochrome oxidase (cox) LAMP primers that amplify a housekeeping gene in plants were used as controls. LAMP assays using a commercially available master mix and a real-time fluorometer allowed detection of the SCWL phytoplasma within 30 minutes at 63oC via the visualization of an amplification plot on screen. The results obtained in this study revealed potential applicability of the LAMP-based assay with 16SrXI SCWL primers for in-field detection of SCWL in comparison with traditional methods that are labour-intensive and prone to cross-contamination during their implementation