Rapid Differentiation of Aspergillus Species from Other Medically Important Opportunistic Molds and Yeasts by PCR-Enzyme Immunoassay

Liliana de Aguirre, Steven F. Hurst, Jong Soo Choi, Jong Hee Shin, Hans Peter Hinrikson,^¶ and Christine J. Morrison^*

Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

Received 6 January 2004/ Returned for modification 20 February 2004/ Accepted 4 May 2004

We developed a PCR-based assay to differentiate medically important species of Aspergillus from one another and from other opportunistic molds and yeasts by employing universal, fungus-specific primers and DNA probes in an enzyme immunoassay format (PCR-EIA). Oligonucleotide probes, directed to the internal transcribed spacer 2 region of ribosomal DNA from Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor, differentiated 41 isolates (3 to 9 each of the respective species; P < 0.001) in a PCR-EIA detection matrix and gave no false-positive reactions with 33 species of Acremonium, Exophiala, Candida, Fusarium, Mucor, Paecilomyces, Penicillium, Rhizopus, Scedosporium, Sporothrix, or other aspergilli tested. A single DNA probe to detect all seven of the most medically important Aspergillus species (A. flavus, A. fumigatus, A. nidulans, A. niger, A. terreus, A. ustus, and A. versicolor) was also designed. Identification of Aspergillus species was accomplished within a single day by the PCR-EIA, and as little as 0.5 pg of fungal DNA could be detected by this system. In addition, fungal DNA extracted from tissues of experimentally infected rabbits was successfully amplified and identified using the PCR-EIA system. This method is simple, rapid, and sensitive for the identification of medically important Aspergillus species and for their differentiation from other opportunistic fungi.

* Corresponding author. Mailing address: Mycotic Diseases Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd., NE, Mailstop G-11, Atlanta, GA 30333. Phone: (404) 639-3098. Fax: (404) 639-3546. E-mail: cjm3@cdc.gov.

Present address: Department of Microbiology, Instituto Investigaciones Veterinarias, Maracay, Venezuela.

Present address: Department of Dermatology, Yeungnam University Medical Center, Taegu City, Korea.

Present address: Department of Laboratory Medicine, Chonnam National University Medical School, Gwanju, Korea.

^¶ Present address: Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.

Journal of Clinical Microbiology, August 2004, p. 3495-3504, Vol. 42, No. 8
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.8.3495-3504.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.