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Loop Mediated Isothermal Amplification Assay for The Detection of Sugarcane White Leaf Disease

Quoc Nguyen: Nong Lam University

<div>Phytoplasma group 16SrXI has been known to be a causal agent of Sugarcane White Leaf Disease (SWLD) resulting in severe yield losses to the sugar industry. Although microscopy, serology based method, dot blot DNA hybridization and PCR have been developed, these techniques require intensive labor, costs, are time-consuming and can only be undertaken in well-equipped labs. Therefore, the objective of this study was to develop a closed tube loop-mediated isothermal amplification (LAMP) assay as an alternative approach for the detection of specific phytoplasmas quickly and efficiently within 30 minutes. Three LAMP primer sets including universal 16S rDNA primers, group XI 16S rDNA primers and plant cytochrome oxidase (COX) gene primers were used to detect phytoplasma DNA sequences and the house keeping gene in plant. LAMP assays using a commercially available master mix and a realtime fluorometer allowed us to detect phytoplasma within 30 minutes at 63<sup>0</sup>C via the visualization of amplification plot and melting curve on screen. The results obtained in this study revealed potential applicability of the closed tube LAMP assay for in- field detection of the phytoplasma causing SWLD in comparison with traditional methods and opened tube LAMP assays which are prone to cross- contamination during the implementation</div>