Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR

Abstract

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.

FIG. 1.

Examples of the serial dilution of DNA amplicons. (A) Primers ITS1-ITS4; (B) primers CGL1-CGL2; (C) primers CTR1-CTR2; (D) primers CPA1-CPA2. Lanes 1 to 6, specific amplicons generated by using 10⁶, 10⁵, 10⁴, 10³, 10², and 10¹ molecules, respectively; lanes 7, negative control, lacking template DNA; lanes M, 100-bp DNA length ladder.

FIG. 2.

Multiplex PCR performed with genomic DNA from two clinical isolates of each species. (Top) Multiplex G-T-P PCR. (Bottom) Multiplex F-A-N PCR. Lanes 1 and 2, C. glabrata; lanes 3 and 4, C. tropicalis; lanes 5 and 6, C. parapsilosis; lanes 7 and 8, A. fumigatus; lanes 9 and 10, C. albicans; lanes 11 and 12, C. neoformans; lane 13, mixed DNA of C. glabrata, C. tropicalis, and C. parapsilosis; lane 14, mixed DNA of A. fumigatus, C. albicans, and C. neoformans; lane 15, negative control without template DNA; lanes M, 100-bp DNA length ladder.

FIG. 3.

Example of multiplex G-T-P (A) and multiplex F-A-N (B) PCRs directly from yeast colonies. The figure shows amplicons from each of five individual clinical isolates of C. glabrata (lanes 1 to 5), C. tropicalis (lanes 6 to 10), C. parapsilosis (lanes 11 to 15), A. fumigatus (lanes 16 to 20), C. albicans (lanes 21 to 25), and C. neoformans (lanes 26 to 30). Lanes 31, mixed DNA of C. glabrata, C. tropicalis, and C. parapsilosis. Lanes 32, mixed DNA of A. fumigatus, C. albicans, and C. neoformans. Lanes M, 50-bp DNA ladder.