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Journal of Clinical Microbiology, August 2002, p. 2860-2865, Vol. 40, No. 8
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.8.2860-2865.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Rapid Identification of Pathogenic Fungi Directly from Cultures by Using Multiplex PCR

Guizhen Luo and Thomas G. Mitchell*

Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710

Received 4 October 2001/ Returned for modification 24 December 2001/ Accepted 8 May 2002

A multiplex PCR method was developed to identify simultaneously multiple fungal pathogens in a single reaction. Five sets of species-specific primers were designed from the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the rRNA gene to identify Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus. Another set of previously published ITS primers, CN4 and CN5, were used to identify Cryptococcus neoformans. Three sets of primers were used in one multiplex PCR to identify three different species. Six different species of pathogenic fungi can be identified with two multiplex PCRs. Furthermore, instead of using templates of purified genomic DNA, we performed the PCR directly from yeast colonies or cultures, which simplified the procedure and precluded contamination during the extraction of DNA. A total of 242 fungal isolates were tested, representing 13 species of yeasts, four species of Aspergillus, and three zygomycetes. The multiplex PCR was tested on isolated DNA or fungal colonies, and both provided 100% sensitivity and specificity. However, DNA from only about half the molds could be amplified directly from mycelial fragments, while DNA from every yeast colony was amplified. This multiplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify common clinical fungal isolates.

* Corresponding author. Mailing address: Box 3803, Department of Microbiology, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-5792. Fax: (919) 681-8911. E-mail: tom.mitchell{at}duke.edu.

Journal of Clinical Microbiology, August 2002, p. 2860-2865, Vol. 40, No. 8
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.8.2860-2865.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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