This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Müller, F.-M. C.
Right arrow Articles by Walsh, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Müller, F.-M. C.
Right arrow Articles by Walsh, T. J.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, June 1998, p. 1625-1629, Vol. 36, No. 6
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Rapid Extraction of Genomic DNA from Medically Important Yeasts and Filamentous Fungi by High-Speed Cell Disruption

Frank-Michael C. Müller,* Katherine E. Werner, Miki Kasai, Andrea Francesconi, Stephen J. Chanock, and Thomas J. Walsh*

Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Received 6 October 1997/Returned for modification 30 October 1997/Accepted 17 March 1998

Current methods of DNA extraction from different fungal pathogens are often time-consuming and require the use of toxic chemicals. DNA isolation from some fungal organisms is difficult due to cell walls or capsules that are not readily susceptible to lysis. We therefore investigated a new and rapid DNA isolation method using high-speed cell disruption (HSCD) incorporating chaotropic reagents and lysing matrices in comparison to standard phenol-chloroform (PC) extraction protocols for isolation of DNA from three medically important yeasts (Candida albicans, Cryptococcus neoformans, and Trichosporon beigelii) and two filamentous fungi (Aspergillus fumigatus and Fusarium solani). Additional extractions by HSCD were performed on Saccharomyces cerevisiae, Pseudallescheria boydii, and Rhizopus arrhizus. Two different inocula (108 and 107 CFU) were compared for optimization of obtained yields. The entire extraction procedure was performed on as many as 12 samples within 1 h compared to 6 h for PC extraction. In comparison to the PC procedure, HSCD DNA extraction demonstrated significantly greater yields for 108 CFU of C. albicans, T. beigelii, A. fumigatus, and F. solani (P <=  0.005), 107 CFU of C. neoformans (P <=  0.05), and 107 CFU of A. fumigatus (P <=  0.01). Yields were within the same range for 108 CFU of C. neoformans and 107 CFU of C. albicans for both HSCD extraction and PC extraction. For 107 CFU of T. beigelii, PC extraction resulted in a greater yield than did HSCD (P <=  0.05). Yields obtained from 108 and 107 CFU were significantly greater for filamentous fungi than for yeasts by the HSCD extraction procedure (P < 0.0001). By the PC extraction procedure, differences were not significant. For all eight organisms, the rapid extraction procedure resulted in good yield, integrity, and quality of DNA as demonstrated by restriction fragment length polymorphism, PCR, and random amplified polymorphic DNA. We conclude that mechanical disruption of fungal cells by HSCD is a safe, rapid, and efficient procedure for extracting genomic DNA from medically important yeasts and especially from filamentous fungi.

* Corresponding author. Mailing address for Thomas J. Walsh: Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute, Building 10, Room 13N240, Bethesda, MD 20892. Phone: (301) 402-0023. Fax: (301) 402-0575. E-mail: walsht{at}pbmac.nci.nih.gov. Mailing address for Frank-Michael C. Müller: Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. Phone: 49-931-312575. Fax: 49-931-312578. E-mail: frank.mueller{at}mail.uni-wuerzburg.de.

Journal of Clinical Microbiology, June 1998, p. 1625-1629, Vol. 36, No. 6
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Kasai, M., Harrington, S. M., Francesconi, A., Petraitis, V., Petraitiene, R., Beveridge, M. G., Knudsen, T., Milanovich, J., Cotton, M. P., Hughes, J., Schaufele, R. L., Sein, T., Bacher, J., Murray, P. R., Kontoyiannis, D. P., Walsh, T. J. (2008). Detection of a Molecular Biomarker for Zygomycetes by Quantitative PCR Assays of Plasma, Bronchoalveolar Lavage, and Lung Tissue in a Rabbit Model of Experimental Pulmonary Zygomycosis. J. Clin. Microbiol. 46: 3690-3702 [Abstract] [Full Text]  
  • Francesconi, A., Kasai, M., Harrington, S. M., Beveridge, M. G., Petraitiene, R., Petraitis, V., Schaufele, R. L., Walsh, T. J. (2008). Automated and Manual Methods of DNA Extraction for Aspergillus fumigatus and Rhizopus oryzae Analyzed by Quantitative Real-Time PCR. J. Clin. Microbiol. 46: 1978-1984 [Abstract] [Full Text]  
  • Metwally, L., Fairley, D. J., Coyle, P. V., Hay, R. J., Hedderwick, S., McCloskey, B., O'Neill, H. J., Webb, C. H., Elbaz, W., McMullan, R. (2008). Improving molecular detection of Candida DNA in whole blood: comparison of seven fungal DNA extraction protocols using real-time PCR. J Med Microbiol 57: 296-303 [Abstract] [Full Text]  
  • Griffiths, L. J., Anyim, M., Doffman, S. R., Wilks, M., Millar, M. R., Agrawal, S. G. (2006). Comparison of DNA extraction methods for Aspergillus fumigatus using real-time PCR.. J Med Microbiol 55: 1187-1191 [Abstract] [Full Text]  
  • Francesconi, A., Kasai, M., Petraitiene, R., Petraitis, V., Kelaher, A. M., Schaufele, R., Hope, W. W., Shea, Y. R., Bacher, J., Walsh, T. J. (2006). Characterization and Comparison of Galactomannan Enzyme Immunoassay and Quantitative Real-Time PCR Assay for Detection of Aspergillus fumigatus in Bronchoalveolar Lavage Fluid from Experimental Invasive Pulmonary Aspergillosis.. J. Clin. Microbiol. 44: 2475-2480 [Abstract] [Full Text]  
  • Fredricks, D. N., Smith, C., Meier, A. (2005). Comparison of Six DNA Extraction Methods for Recovery of Fungal DNA as Assessed by Quantitative PCR. J. Clin. Microbiol. 43: 5122-5128 [Abstract] [Full Text]  
  • de Aguirre, L., Hurst, S. F., Choi, J. S., Shin, J. H., Hinrikson, H. P., Morrison, C. J. (2004). Rapid Differentiation of Aspergillus Species from Other Medically Important Opportunistic Molds and Yeasts by PCR-Enzyme Immunoassay. J. Clin. Microbiol. 42: 3495-3504 [Abstract] [Full Text]  
  • O'Sullivan, C. E., Kasai, M., Francesconi, A., Petraitis, V., Petraitiene, R., Kelaher, A. M., Sarafandi, A. A., Walsh, T. J. (2003). Development and Validation of a Quantitative Real-Time PCR Assay Using Fluorescence Resonance Energy Transfer Technology for Detection of Aspergillus fumigatus in Experimental Invasive Pulmonary Aspergillosis. J. Clin. Microbiol. 41: 5676-5682 [Abstract] [Full Text]  
  • Loeffler, J., Schmidt, K., Hebart, H., Schumacher, U., Einsele, H. (2002). Automated Extraction of Genomic DNA from Medically Important Yeast Species and Filamentous Fungi by Using the MagNA Pure LC System. J. Clin. Microbiol. 40: 2240-2243 [Abstract] [Full Text]  
  • Zhao, J., Kong, F., Li, R., Wang, X., Wan, Z., Wang, D. (2001). Identification of Aspergillus fumigatus and Related Species by Nested PCR Targeting Ribosomal DNA Internal Transcribed Spacer Regions. J. Clin. Microbiol. 39: 2261-2266 [Abstract] [Full Text]  
  • Loeffler, J., Hebart, H., Bialek, R., Hagmeyer, L., Schmidt, D., Serey, F.-P., Hartmann, M., Eucker, J., Einsele, H. (1999). Contaminations Occurring in Fungal PCR Assays. J. Clin. Microbiol. 37: 1200-1202 [Abstract] [Full Text]  
  • Rimek, D., Garg, A. P., Haas, W. H., Kappe, R. (1999). Identification of Contaminating Fungal DNA Sequences in Zymolyase. J. Clin. Microbiol. 37: 830-831 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»