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Loop-Mediated Isothermal Amplification-Based Detection of Fusarium graminearum

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Fungal Diagnostics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 968))

Abstract

Molecular biological detection and quantification of fungal DNA targets today relies mainly on the ­application of the polymerase chain reaction (PCR). However, this well-recognized technique necessitates the use of highly purified DNA, in a well-equipped lab environment by trained personnel. The method has therefore considerable restrictions when it comes to on-site testing applications for phytopathogenic, mycotoxigenic, and medically relevant fungi and yeasts. As opposed to PCR, molecular biological detection of fungal DNA with isothermal amplification methods is performed at constant temperature. This makes thermal cycling superfluous and enables application of molecular detection assays on site. Moreover, detection of amplification product is done in-tube with no post-amplification manipulations necessary. Here we describe the use of loop-mediated isothermal amplification (LAMP) with indirect in-tube detection of DNA amplification as a rapid and robust method. Detection of F. graminearum in pure cultures and in cereal samples will be described as an example for the high potential which LAMP may hold for future developments in fungal detection assays.

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References

  1. Mullis KB, Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335–350

    Article  PubMed  CAS  Google Scholar 

  2. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Hguchi R, Horn GT, Mullis KB, Ehrlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491

    Article  PubMed  CAS  Google Scholar 

  3. Niessen L (2008) PCR-based diagnosis and quantification of mycotoxin-producing fungi. Adv Food Nutr Res 54:81–137

    Article  PubMed  CAS  Google Scholar 

  4. 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

    Article  PubMed  CAS  Google Scholar 

  5. Nagamine K, Hase T, Notomi T (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16:223–229

    Article  PubMed  CAS  Google Scholar 

  6. Njiru ZK, Mikosza ASJ, Matovu E, Enyaru JCK, Ouma JO, Kibona SN, Thompson RCA, Ndung’u JM (2008) African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA. Int J Parasitol 38:589–599

    Article  PubMed  CAS  Google Scholar 

  7. Mori Y, Hirano T, Notomi T (2006) Sequence specific visual detection of LAMP reactions by addition of cationic polymers. BMC Biotechnol 6(3):10

    Google Scholar 

  8. 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 289:150–154

    Article  PubMed  CAS  Google Scholar 

  9. Tomita N, Mori Y, Kanda H, Notomi T (2008) Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3:877–882

    Article  PubMed  CAS  Google Scholar 

  10. Mori Y, Kitao M, Tomita N, Notomi T (2004) Real-time turbidimetry of LAMP reaction for quantifying template DNA. J Biochem Biophys Methods 59:145–157

    Article  PubMed  CAS  Google Scholar 

  11. Endo S, Komori T, Ricci G, Sano A, Yokoyama K, Ohori A, Kamai K, Franco M, Miyaji M, Nishimura K (2004) Detection of gp43 of Paracoccidioides brasiliensis by the loop-mediated isothermal amplification (LAMP) method. FEMS Microbiol Lett 234:93–97

    Article  PubMed  CAS  Google Scholar 

  12. Ohori A, Endo Sano A, Yokoyama K, Yarita K, Yamaguchi M, Kamai K, Miyaji M, Nishimura K (2006) Rapid identification of Ochroconis gallopava by a loop-mediated isothermal amplification (LAMP) method. Vet Microbiol 114:359–365

    Article  PubMed  CAS  Google Scholar 

  13. Tomlinson JA, Barker I, Boonham N (2007) Faster, simpler, more-specific methods for improved molecular detection of Phytophthora ramorum in the field. Appl Environ Microbiol 73:4040–4047

    Article  PubMed  CAS  Google Scholar 

  14. Hayashi N, Arai R, Tada S, Taguchi H, Ogawa Y (2007) Detection and identification of Brettanomyces/Dekkera sp. yeasts with a loop-mediated isothermal amplification method. Food Microbiol 24:778–785

    Article  PubMed  CAS  Google Scholar 

  15. Gadkar V, Rillig MC (2008) Evaluation of loop-mediated isothermal amplification (LAMP) to rapidly detect arbuscular mycorrhizal fungi. Soil Biol Biochem 40:540–543

    Article  CAS  Google Scholar 

  16. Sun J, Najafzadeh MJ, Vicente V, Xi L, de Hoog GS (2010) Rapid detection of pathogenic fungi using loop-mediated isothermal amplification, exemplified by Fonsecaea agents of chromoblastomycosis. J Microbiol Meth 80:19–24

    PubMed  CAS  Google Scholar 

  17. Sun J, Li X, Zeng H, Zhi Z, Lu C, Xi L, de Hoog GS (2010) Development and evaluation of loop-mediated isothermal amplification(LAMP) for the rapid diagnosis of Penicillium marneffei in archived tissue samples. FEMS Immunol Med Microbiol 58:381–388

    PubMed  CAS  Google Scholar 

  18. Lucas S, da Luz Martins M, Flores O, Meyer W, Spencer-Martins I, Inácio J (2010) Differentiation of Cryptococcus neoformans varieties and Cryptococcus gattii using CAP59-based loop-mediated isothermal DNA amplification. Clin Microbiol Infect 16:711–714

    Article  PubMed  CAS  Google Scholar 

  19. Matsuzawa T, Tanaka R, Horie Y, Gonoi T, Yaguchi T (2010) Development of rapid and specific molecular discrimination methods fro pathogenic Emericella species. Nihon Ishinkin Gakkai Zasshi 51:109–116

    Article  PubMed  CAS  Google Scholar 

  20. Tomlinson JA, Dickinson MJ, Boonham N (2010) Detection of Botrytis cinerea by loop-mediated isothermal amplification. Lett Appl Microbiol 51:650–657

    Article  PubMed  CAS  Google Scholar 

  21. Niessen L, Vogel RF (2010) Detection of Fusarium graminearum DNA using a loop-mediated isothermal amplification (LAMP) assay. Int J Food Microbiol 140:183–191

    Article  PubMed  CAS  Google Scholar 

  22. Doohan FM, Brennan J, Cook BM (2003) Influence of climatic factors on Fusarium species pathogenic to cereals. Eur J Plant Pathol 109:755–768

    Article  Google Scholar 

  23. Hippeli S, Elstner EF (2002) Are hydrophobins and/or non specific lipid transfer proteins (ns-LTPs) responsible for gushing in beer? New hypotheses on the chemical nature of gushing inducing factors. Z Naturforsch C 57:1–8

    PubMed  CAS  Google Scholar 

  24. Nirenberg H (1976) Untersuchungen über die morphologische und biologische Differenzierung in der Fusarium-Sektion Liseola. Mitt Biolog Bundesanstalt für Land-u Forstw (Berlin-Dahlem) 169:117

    Google Scholar 

  25. Lee SB, Taylor JW (1990) Isolation of DNA from fungal mycelia and single spores. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, New York, pp 282–287

    Google Scholar 

  26. Hatano B, Maki T, Obara T, Fukumoto H, Hagisawa K, Matsushita Y, Okutani A, Bazartseren B, Inoue S, Sata T, Katano H (2010) LAMP using a disposable pocket warmer for anthrax detection, a highly mobile and reliable method for anti-bioterrorism. Jpn J Infect Dis 63:36–40

    PubMed  CAS  Google Scholar 

  27. Niessen L, Gräfenhan T, Vogel RF (2012) ATP citrate lyase 1 (acl1) gene-based loop-mediated isothermal amplification assay for the detection of the Fusarium tricinctum species complex in pure cultures and in cereal samples. Int J Food Microbiol 158:171–185

    Google Scholar 

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Authors and Affiliations

  1. Weihenstephan Center of Food and Life Sciences, TU-München, Lehrstuhl für Technische Mikrobiologie, Freising, Germany

    Ludwig Niessen

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  1. Ludwig Niessen
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Correspondence to Ludwig Niessen .

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Editors and Affiliations

  1. , NCBES, Molecular Diagnostics Research Gr, National University of Ireland, Galway, IDA Business Park, Dangan, Galway, Ireland

    Louise O'Connor

  2. , NCBES, Molecular Diagnostics Research Gr, National University of Ireland, Galway, IDA Business Park, Dangan, Galway, Ireland

    Barry Glynn

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Niessen, L. (2013). Loop-Mediated Isothermal Amplification-Based Detection of Fusarium graminearum . In: O'Connor, L., Glynn, B. (eds) Fungal Diagnostics. Methods in Molecular Biology, vol 968. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-257-5_14

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  • DOI: https://doi.org/10.1007/978-1-62703-257-5_14

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-256-8

  • Online ISBN: 978-1-62703-257-5

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