This Article Full Text Full Text (PDF) Other Versions of this Article: JCM.02246-07v1 46/6/1978    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Francesconi, A. Articles by Walsh, T. J. Search for Related Content PubMed PubMed Citation Articles by Francesconi, A. Articles by Walsh, T. J.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, June 2008, p. 1978-1984, Vol. 46, No. 6
0095-1137/08/$08.00+0     doi:10.1128/JCM.02246-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Automated and Manual Methods of DNA Extraction for Aspergillus fumigatus and Rhizopus oryzae Analyzed by Quantitative Real-Time PCR

National Cancer Institute, National Institutes of Health, Bethesda, Maryland,¹ NIH Clinical Center, NIH, Bethesda, Maryland,² LASP, SAIC-Frederick Inc., Frederick, Maryland³

Received 20 November 2007/ Returned for modification 31 December 2007/ Accepted 4 March 2008

Quantitative real-time PCR (qPCR) may improve the detection of fungal pathogens. Extraction of DNA from fungal pathogens is fundamental to optimization of qPCR; however, the loss of fungal DNA during the extraction process is a major limitation to molecular diagnostic tools for pathogenic fungi. We therefore studied representative automated and manual extraction methods for Aspergillus fumigatus and Rhizopus oryzae. Both were analyzed by qPCR for their ability to extract DNA from propagules and germinated hyphal elements (GHE). The limit of detection of A. fumigatus and R. oryzae GHE in bronchoalveolar lavage (BAL) fluid with either extraction method was 1 GHE/ml. Both methods efficiently extracted DNA from A. fumigatus, with a limit of detection of 1 x 10² conidia. Extraction of R. oryzae by the manual method resulted in a limit of detection of 1 x 10³ sporangiospores. However, extraction with the automated method resulted in a limit of detection of 1 x 10¹ sporangiospores. The amount of time to process 24 samples by the automated method was 2.5 h prior to transferring for automation, 1.3 h of automation, and 10 min postautomation, resulting in a total time of 4 h. The total time required for the manual method was 5.25 h. The automated and manual methods were similar in sensitivity for DNA extraction from A. fumigatus conidia and GHE. For R. oryzae, the automated method was more sensitive for DNA extraction of sporangiospores, while the manual method was more sensitive for GHE in BAL fluid.

Published ahead of print on 19 March 2008.

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]