Abstract
The rapid and inexpensive extraction of fungal genomic DNA that is of sufficient quality for molecular approaches is central to the molecular identification, epidemiological analysis, taxonomy, and strain typing of pathogenic fungi. Although many commercially available and in-house extraction procedures do eliminate the majority of contaminants that commonly inhibit molecular approaches, the inherent difficulties in breaking fungal cell walls lead to protocols that are labor intensive and that routinely take several hours to complete. Here we describe several methods that we have developed in our laboratory that allow the extremely rapid and inexpensive preparation of fungal genomic DNA.
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Nucci M, Marr KA (2005) Emerging fungal diseases. Clin Infect Dis 41:521–526
Richardson M, Lass-Flörl C (2008) Changing epidemiology of systemic fungal infections. Clin Microbiol Infect 14(Suppl 4):5–24
Linton CJ, Borman AM, Cheung G, Holmes AD, Szekely A, Palmer MD, Bridge PD, Campbell CK, Johnson EM (2007) Molecular identification of unusual pathogenic yeast isolates by Large Ribosomal Subunit gene sequencing: 2 years experience at the UK Mycology Reference Laboratory. J Clin Microbiol 45:1152–1158
Borman AM, Linton CJ, Miles S-J, Johnson EM (2008) Molecular identification of pathogenic fungi. J Antimicrob Chemother 61:i7–i12
Tavanti A, Davidson AD, Gow NAR, Maiden MCJ, Odds FC (2005) Candida orthopsilosis and Candida metapsilosis spp. nov. To replace Candida parapsilosis groups II and III. J Clin Microbiol 43:284–292
Gilgado F, Cano J, Gené J, Guarro J (2005) Molecular phylogeny of the Pseudallescheria boydii species complex: proposal of two new species. J Clin Microbiol 43:4930–4942
O’Donnell K, Sutton DA, Fothergill A, McCarthy D, Rinaldi MG, Brandt ME, Zhang N, Geiser DM (2008) Molecular phylogenetic diversity, multilocus haplotype nomenclature, and in vitro antifungal resistance within the Fusarium solani species complex. J Clin Microbiol 46:2477–2490
Balajee SA, Kano R, Baddley JW, Moser SA, Marr KA, Alexander BD, Andes D, Kontoyiannis DP, Perrone G, Peterson S, Brandt ME, Pappas PG, Chiller T (2009) Molecular identification of Aspergillus species collected from the transplant-associated surveillance network. J Clin Microbiol 47:3138–3141
Balajee SA, Weaver M, Imhof A, Gribskov J, Marr KA (2004) Aspergillus fumigatus variant with decreased susceptibility to multiple antifungals. Antimicrob Agents Chemother 48:1197–1203
Balajee SA, Gribskov J, Brandt M, Ito J, Fothergill A, Marr KA (2005) Mistaken identity: Neosartorya pseudofischeri and its anamorph masquerading as Aspergillus fumigatus. J Clin Microbiol 43:5996–5999
Alcazar-Fuoli L, Mellado E, Alastruey-Izquierdo A, Cuenca-Estrella M, Rodriguez-Tudela JL (2008) Aspergillus section Fumigati: antifungal susceptibility patterns and sequence-based identification. Antimicrob Agents Chemother 52:1244–1251
Borman AM, Petch R, Linton CJ, Palmer MD, Bridge PD, Johnson EM (2008) Candida nivariensis, an emerging pathogenic yeast with multi-drug resistance to antifungal agents. J Clin Microbiol 46:933–938
Houbraken J, Verweij PE, Rijs AJMM, Borman AM, Samson RA (2010) Identification of Paecilomyces variotii in clinical samples and settings. J Clin Microbiol 48:2754–2761
Tang C, Holden D, Aufauvre-Brown A, Cohen J (1993) The detections of Aspergillus spp. by the polymerase chain reaction and its evaluation of bronchealveolar lavage fluid. Am Rev Respir Dis 148:1313–1317
Al-Samarrai TH, Schmid J (2000) A simple method for extraction of fungal genomic DNA. Lett Appl Microbiol 30:53–56
Cenis JL (1992) Rapid extraction of fungal DNA for PCR amplification. Nucleic Acids Res 20:2380
Dean TR, Betancourt D, Menetrez MY (2004) A rapid DNA extraction method for PCR identification of fungal indoor air contaminants. J Microbiol Methods 56:431–434
Faggi E, Pini G, Campisi E (2005) Use of magnetic beads to extract fungal DNA. Mycoses 48:3–7
Guo JR, Schneider F, Abd-Elsalam KA, Vereet JA (2005) Rapid and efficient extraction of genomic DNA from different phytopathogenic fungi using DNAzol reagent. Biotechnol Lett 27:3–6
Hopfer RL, Walden P, Setterquist S, Highsmith WE (1993) Detection and differentiation of fungi in clinical specimens using polymerase chain recation amplification and restriction enzyme analysis. J Med Vet Mycol 31:65–75
Jin J, Lee Y-K, Wickes BL (2004) Simple chemical extraction method for DNA isolation from Aspergillus fumigatus and other Aspergillus species. J Clin Microbiol 42:4293–4296
Muller F-MC, Werner KE, Kasai M, Francesconi A, Chanock SJ, Walsh TJ (1998) Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption. J Clin Microbiol 36:1625–1629
Van Burik JA, Schreckhise RW, White TC, Bowden RA, Myerson D (1998) Comparison of six extraction techniques for isolation of DNA from filamentous fungi. Med Mycol 36:299–303
Liu D, Coloe S, Baird R, Pederson J (2000) Application of PCR to the identification of dermatophyte fungi. J Med Microbiol 49:493–497
Borman AM, Linton CJ, Miles S-J, Campbell CK, Johnson EM (2006) Ultra-rapid preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter paper technology—a re-usable DNA archiving system. Med Mycol 44:389–398
Borman AM, Fraser M, Linton CJ, Palmer MD, Johnson EM (2010) An improved method for the preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter papers. Mycopathologia 169:445–449
Acknowledgments
We thank the other staff of the UK National Mycology Reference Laboratory for their interest in these approaches and for providing much of the data on which these approaches are based. We are also indebted to Whatman International for supplying the FTA cards and wash reagents used for these studies and to Haroun Shah and Dunstan Rajendram for their invaluable advice on FTA applications.
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Borman, A.M., Palmer, M., Johnson, E.M. (2013). Rapid Methods for the Extraction and Archiving of Molecular Grade Fungal Genomic DNA. 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_3
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DOI: https://doi.org/10.1007/978-1-62703-257-5_3
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