Rapid identification of fungi by using the ITS2 genetic region and an automated fluorescent capillary electrophoresis system

Abstract

Invasive fungal disease often plays an important role in the morbidity and mortality of immunocompromised patients. The poor sensitivity of current fungal blood culture and histological practices has led to the development of highly sensitive and specific molecular techniques, such as the PCR. Sequence variability of the internal transcribed spacer 2 (ITS2) region of fungi is potentially useful in rapid and accurate diagnosis of clinical fungal isolates. PCR with fungus-specific primers targeted toward conserved sequences of the 5.8S and 28S ribosomal DNA (rDNA) results in amplification of the species-specific ITS2 regions, which are variable in amplicon length. We have made use of the ABI PRISM 310 genetic analyzer and the ABI PRISM 310 GeneScan analysis software for the determination of variable size differences of the ITS2 region of clinically important fungi, including Candida and non-Candida yeasts, Aspergillus species, and a variety of dermatophytes. No cross-reaction occurred when samples were tested against human and bacterial genomic DNA. We have found that most clinically significant fungal isolates can be differentiated by this method, and it therefore serves to be a promising tool for the rapid (<7 h) diagnosis of fungemia and other invasive fungal infections.

FIG. 1

Schematic representation of the fungal ribosomal genes containing the primer target areas used in the amplification of the ITS2 region.

FIG. 2

Specificity of universal ITS2 primers against bacteria and human genomic DNA. PCR amplification using the ITS4 and ITS86 primer pair was performed as described in Materials and Methods. The following DNA templates were used for PCR (by lane): 1, Staphylococcus epidermidis ATCC 12228; 2, Escherichia coli ATCC 25922; 3, Staphylococcus aureus ATCC 25923; 4, Pseudomonas aeruginosa ATCC 27853; 5, Clostridium perfringens ATCC 13124; 6, human whole blood; 7, human leukocytes; 8, human liver; 9, Candida albicans ATCC 10231; and 10, H₂O contamination control.

FIG. 3

Detection of fungal species by agarose gel electrophoresis. PCR amplification of variable Candida species from culture colonies using the ITS4 and ITS86 universal fungal primers was performed as described in Materials and Methods. Lanes: L, 123-bp ladder; 1, Candida albicans; 2, Candida kefyr; 3, Candida zeylanoides; 4, Candida tropicalis; 5, Candida krusei; 6, Candida glabrata; 7, Candida guilliermondii; 8, Candida lusitaniae; 9, Candida parapsilosis.

FIG. 4

Electropherograms of five Candida species as analyzed by the ABI PRISM 310 genetic analyzer. Control strains (graphs 1 to 5) were amplified by using ITS4 and fluorescently labeled ITS86 and dUTP as described in Materials and Methods. Each was run separately on the capillary electrophoresis system along with an internal size standard (GeneScan ROX-500). Standard peaks are shown as a separate electropherogram (graph 6) for clarity of illustration. The standard peak sizes are 139, 150, 150, 200, 240, 300, 340, 350, and 400 bp. Graphs: 1, Candida albicans ATCC 10231; 2, Candida tropicalis ATCC 66029; 3, Candida glabrata ATCC 90030; 4, Candida lusitaniae ATCC 42720; 5, Candida krusei ATCC 6258.