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Detection of Puccinia kuehnii Causing Sugarcane Orange Rust with a Loop-Mediated Isothermal Amplification-Based Assay

Chandra, Amaresh, Keizerweerd, Amber T., Grisham, Michael P.
Molecular biotechnology 2016 v.58 no.3 pp. 188-196
DNA, DNA primers, Puccinia kuehnii, Puccinia melanocephala, color, disease detection, dyes, fungi, leaf rust, loop-mediated isothermal amplification, magnesium sulfate, nucleotide sequences, pathogens, quantitative polymerase chain reaction, rapid methods, sugarcane
Puccinia kuehnii is a fungal pathogen that causes orange rust in sugarcane, which is now prevalent in many countries. At the early stage of disease, it is almost indistinguishable from brown rust, which is caused by Puccinia melanocephala. Although several PCR assays are available to detect these diseases, the loop-mediated isothermal amplification (LAMP)-based assay has been reported to be more economical and easier to perform. Under isothermal conditions, DNA is amplified with high specificity and rapidity. Moreover, visual judgment of color change without further post-amplification processing makes the method convenient. The present study was undertaken to detect P. kuehnii genomic DNA using four primers corresponding to a unique DNA sequence of P. kuehnii. The LAMP assay was found to be optimal when 8 mM MgSO₄ was used and the reaction was incubated at 63 °C for 90 min. Positive samples showed a color change from orange to green upon SYBR Green I dye addition. Specificity of the LAMP test was checked with DNA of P. melanocephala, which showed no reaction. Sensitivity of the LAMP method was observed to be the same as real-time PCR at 0.1 ng, thus providing a rapid and more affordable option for early disease detection.