JCM Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Other Versions of this Article:
JCM.02246-07v1
46/6/1978    most recent
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
Google Scholar
Right arrow Articles by Francesconi, A.
Right arrow Articles by Walsh, T. J.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Francesconi, A.
Right arrow 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{triangledown}

Andrea Francesconi,1 Miki Kasai,1 Susan M. Harrington,2 Mara G. Beveridge,1 Ruta Petraitiene,1,3 Vidmantas Petraitis,1,3 Robert L. Schaufele,1 and Thomas J. Walsh1*

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.

* Corresponding author. Mailing address: Immunocompromised Host Section, Pediatric Oncology Branch, National Cancer Institute, Bldg. 10-CRC, Rm. 1-5740, Bethesda, MD 20892. Phone: (301) 402-0023. Fax: (301) 480-2308. E-mail: walsht{at}mail.nih.gov

{triangledown} Published ahead of print on 19 March 2008.

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.





Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Antimicrob. Agents Chemother. Clin. Microbiol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS

Copyright © 2008 by the American Society for Microbiology. All rights reserved.