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Establishment of a loop-mediated isothermal amplification (LAMP) assay for the detection of phytoplasma-associated cassava witches’ broom disease

An Erratum to this article was published on 28 March 2016

Abstract

Cassava (Manihot esculenta Crantz) is one of the most important food crops in the tropics; however, bacterial phytopathogens pose a serious threat to its farming. Cassava Witches’ Broom Disease (CWB) is caused by the infection of phytoplasma and is manifested as reduction in tuber yield and starch content at harvest of 10 and 30 %, respectively. Although polymerase-chain reaction provides the gold standard in diagnostics, this method requires significant investments in infrastructure and training. Here, we developed a loop-mediated isothermal amplification (LAMP) assay that allows specific detection of phytoplasma from field-collected samples. Three primer sets were designed, of which two detected phytoplasma DNA sequence encoding 16S rRNA (16S rDNA), the other detected cassava actin. Following a 1 h LAMP reaction at 63 °C, a positive reaction can be visualized by agarose gel electrophoresis, hydroxynaphthol blue color change, or the presence of a precipitate. In a pilot field study, the assay was able to rapidly distinguish between healthy and CWB-infected cassava. With further development, a LAMP for routine on-site screening of cassava crops can be envisioned.

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Acknowledgments

DTL receives funding from the National Foundation for Science and Technology Development (NAFOSTED) under Grant Number 106-NN.02-2013.46. The authors also would like to acknowledge a support from the EC and the International Fund for Agriculture Development (IFAD) to the International Center for Tropical Agriculture (CIAT) and its partners. The work was conducted at the International Laboratory for Cassava Molecular Breeding (ILCMB) with access to equipment invested by the CGIAR-RTB program. We thank Inge Seim and Georgina Smith for correcting English usage in this manuscript.

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Correspondence to Dung Tien Le.

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Vu, N.T., Pardo, J.M., Alvarez, E. et al. Establishment of a loop-mediated isothermal amplification (LAMP) assay for the detection of phytoplasma-associated cassava witches’ broom disease. Appl Biol Chem 59, 151–156 (2016). https://doi.org/10.1007/s13765-015-0134-7

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Keywords

  • Cassava
  • Cassava witches’ broom disease
  • Loop-mediated amplification
  • Loop-mediated isothermal amplification
  • Phytoplasma