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Journal of Clinical Microbiology, July 1998, p. 2057-2062, Vol. 36, No. 7
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Utility of Random Amplified Polymorphic DNA PCR and TaqMan Automated Detection in Molecular Identification of Aspergillus fumigatus

Mary E. Brandt,1,* Arvind A. Padhye,1 Leonard W. Mayer,1 and Brian P. Holloway2

Division of Bacterial and Mycotic Diseases1 and Biotechnology Core Facility,2 National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333

Received 6 November 1997/Returned for modification 9 January 1998/Accepted 8 April 1998

We developed a method for the identification of Aspergillus fumigatus fungal isolates by using random amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) cloning and the TaqMan LS50B fluorogenic detection system (Perkin-Elmer Corp., Applied Biosystems, Foster City, Calif.). DNA from seven clinically important Aspergillus species was screened by RAPD-PCR to identify section- or species-specific amplicons. With the OPZ19 RAPD primer a 1,264-bp product was amplified from all A. fumigatus strains initially examined but not from other species. A partial DNA sequence of this product was used to design a specific primer pair, which generated a single 864-bp fragment with DNA from 90 of 100 A. fumigatus isolates when a "touchdown" (65right-arrow55°C) annealing protocol was used. The TaqMan system, a fluorogenic assay which uses the 5'right-arrow3' endonuclease activity of Taq DNA polymerase, detected this 864-bp product with DNA from 89 of these 90 A. fumigatus strains; 1 DNA sample generated an indeterminate result. With DNA from three morphologically typical A. fumigatus isolates, six white ("albino") A. fumigatus isolates, and five of six Neosartorya species (non-A. fumigatus members of the section Fumigati), the 864-bp product was amplified differentially at an annealing temperature of 56°C but not with the touchdown annealing format. No amplicon was detected with DNA from 56 isolates of heterologous Aspergillus, Penicillium, and Paecilomyces species or from Neosartorya fennelliae; TaqMan assay results were either negative (51 isolates) or indeterminate (5 isolates) for all isolates. This RAPD-PCR and TaqMan assay offers promise as a nucleic acid-based system that can be used for the identification of filamentous fungal isolates and that requires no postamplification sample manipulations.

* Corresponding author. Mailing address: Mycotic Diseases Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd., Mailstop D-11, Atlanta, GA 30333. Phone: (404) 639-2842. Fax: (404) 639-4421. E-mail: mbb4{at}cdc.gov.

Journal of Clinical Microbiology, July 1998, p. 2057-2062, Vol. 36, No. 7
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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