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
Mullis KB, Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335–350
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
Niessen L (2008) PCR-based diagnosis and quantification of mycotoxin-producing fungi. Adv Food Nutr Res 54:81–137
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
Nagamine K, Hase T, Notomi T (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16:223–229
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
Mori Y, Hirano T, Notomi T (2006) Sequence specific visual detection of LAMP reactions by addition of cationic polymers. BMC Biotechnol 6(3):10
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
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
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
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
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
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
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
Gadkar V, Rillig MC (2008) Evaluation of loop-mediated isothermal amplification (LAMP) to rapidly detect arbuscular mycorrhizal fungi. Soil Biol Biochem 40:540–543
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
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
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
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
Tomlinson JA, Dickinson MJ, Boonham N (2010) Detection of Botrytis cinerea by loop-mediated isothermal amplification. Lett Appl Microbiol 51:650–657
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
Doohan FM, Brennan J, Cook BM (2003) Influence of climatic factors on Fusarium species pathogenic to cereals. Eur J Plant Pathol 109:755–768
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
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
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
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
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
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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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