Applied Biological Chemistry

, Volume 59, Issue 2, pp 151–156 | Cite as

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

  • Nam Tuan Vu
  • Juan Manuel Pardo
  • Elizabeth Alvarez
  • Ham Huy Le
  • Kris Wyckhuys
  • Kim-Lien Nguyen
  • Dung Tien LeEmail author


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.


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



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.


  1. Alvarez E, Manuel PJ, Fernando MJ, Assunta B, Duc TN, Xuan HT (2013) Detection and identification of ‘Candidatus Phytoplasma asteris’-related phytoplasmas associated with a witches’ broom disease of cassava in Vietnam. Phytopathogenic Mollicutes 3:77–81CrossRefGoogle Scholar
  2. Bekele B, Hodgetts J, Tomlinson J, Boonham N, Nikolić P, Swarbrick P, Dickinson M (2011) Use of a real-time LAMP isothermal assay for detecting 16SrII and XII phytoplasmas in fruit and weeds of the Ethiopian Rift Valley. Plant Pathol 60:345–355CrossRefGoogle Scholar
  3. Bhat AI, Siljo A, Deeshma KP (2013) Rapid detection of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) by loop-mediated isothermal amplification (LAMP). J Virol Methods 193:190–196CrossRefGoogle Scholar
  4. Doi Y, Teranaka M, Yora K, Asuyama H (1967) Mycoplasma or PLT grouplike microrganisms found in the phloem elements of plants infected with mulberry dwarf, potato witches’ broom, aster yellows or pawlonia witches’ broom. Ann Phytopathol Soc Jpn 33:7CrossRefGoogle Scholar
  5. Flôres D, Haas I, Canale M, Bedendo I (2013) Molecular identification of a 16SrIII-B phytoplasma associated with cassava witches’ broom disease. Eur J Plant Pathol 137:237–242CrossRefGoogle Scholar
  6. Goto M, Honda E, Ogura A, Nomoto A, Ken-Ichi Hanaki DVM (2009) Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques 46:167–172CrossRefGoogle Scholar
  7. Hadidi A, Czosnek H, Barba M (2004) DNA microarrays and their potential applications for the detection of plant viruses, viroids, and phytoplasmas. J Plant Pathol 86:97–104Google Scholar
  8. IRPCM (2004) ‘Candidatus Phytoplasma’, a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects. Int J Syst Evol Microbiol 54:1243–1255CrossRefGoogle Scholar
  9. Le DT, Netsu O, Uehara-Ichiki T, Shimizu T, Choi I-R, Omura T, Sasaya T (2010) Molecular detection of nine rice viruses by a reverse-transcription loop-mediated isothermal amplification assay. J Virol Methods 170:90–93CrossRefGoogle Scholar
  10. Le TH, Nguyen NTB, Truong NH, De NV (2012) Development of mitochondrial loop-mediated isothermal amplification for detection of the small Liver Fluke Opisthorchis viverrini (Opisthorchiidae; Trematoda; Platyhelminthes). J Clin Microbiol 50:1178–1184CrossRefGoogle Scholar
  11. Li R, Ling K-S (2014) Development of reverse transcription loop-mediated isothermal amplification assay for rapid detection of an emerging potyvirus: tomato necrotic stunt virus. J Virol Methods 200:35–40CrossRefGoogle Scholar
  12. Li W, Hartung JS, Levy L (2007) Evaluation of DNA amplification methods for improved detection of “Candidatus liberibacter species” associated with Citrus Huanglongbing. Plant Dis 91:51–58CrossRefGoogle Scholar
  13. Mondal KK, Shanmugam V (2013) Advancements in the diagnosis of bacterial plant pathogens: an overview. Biotechnol Mol Biol Rev 8:1–11CrossRefGoogle Scholar
  14. Mori Y, Nagamine K, Tomita N, Notomi T (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 289:150–154CrossRefGoogle Scholar
  15. Nagamine K, Hase T, Notomi T (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16:223–229CrossRefGoogle Scholar
  16. Nguyen TD, Mai QV, Ngo BG, Nguyen HH, Ha CV, Trinh HX (2014) Biological characteristics of cassava witches’ broom disease related to phytoplasma in Dongnai Province. J Sci Dev 12:325–333Google Scholar
  17. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:e63–e63CrossRefGoogle Scholar
  18. Parmessur Y, Aljanabi S, Saumtally S, Dookun-Saumtally A (2002) Sugarcane yellow leaf virus and sugarcane yellows phytoplasma: elimination by tissue culture. Plant Pathol 51:561–566CrossRefGoogle Scholar
  19. Ravindran A, Levy J, Pierson E, Gross DC (2012) Development of a loop-mediated isothermal amplification procedure as a sensitive and rapid method for detection of ‘candidatus liberibacter solanacearum’ in potatoes and Psyllids. Phytopathology 102:899–907CrossRefGoogle Scholar
  20. Razin S, Yogev D, Naot Y (1998) Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 62:1094–1156Google Scholar
  21. Tomlinson JA, Boonham N, Dickinson M (2010) Development and evaluation of a one-hour DNA extraction and loop-mediated isothermal amplification assay for rapid detection of phytoplasmas. Plant Pathol 59:465–471CrossRefGoogle Scholar
  22. Torres E, Bertolini E, Cambra M, Montón C, Martín MP (2005) Real-time PCR for simultaneous and quantitative detection of quarantine phytoplasmas from apple proliferation (16SrX) group. Mol Cell Probes 19:334–340CrossRefGoogle Scholar
  23. Weintraub PG, Beanland L (2006) Insect vectors of phytoplasmas. Annu Rev Entomol 51:91–111CrossRefGoogle Scholar

Copyright information

© The Korean Society for Applied Biological Chemistry 2016

Authors and Affiliations

  • Nam Tuan Vu
    • 1
  • Juan Manuel Pardo
    • 2
  • Elizabeth Alvarez
    • 2
  • Ham Huy Le
    • 1
  • Kris Wyckhuys
    • 2
  • Kim-Lien Nguyen
    • 1
  • Dung Tien Le
    • 1
    Email author
  1. 1.International Laboratory for Cassava Molecular Breeding (ILCMB), National Key Laboratory of Plant and Cell Technology, Agricultural Genetics Institute (AGI)Vietnam Academy of Agricultural Science (VAAS)HanoiVietnam
  2. 2.International Center for Tropical Agriculture (CIAT)CaliColombia

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