MICROTITRATION PLATE ENZYME-IMMUNOASSAY TO DETECT PCR-AMPLIFIED DNA FROM CANDIDA SPECIES IN BLOOD

We developed a microtitration plate enzyme immunoassay to detect PCR-a mplified DNA from Candida species. Nucleotide sequences derived from t he internal transcribed spacer (ITS) region of fungal rDNA were used t o develop species-specific oligonucleotide probes for Candida albicans , C. tropicalis, C. parapsilosis, and C. krusei. No cross-hybridizatio n was detected with any other fungal, bacterial, or human DNAs tested. In contrast, a C. (Torulopsis) glabrata probe cross-reacted with Sacc haromyces cerevisiae DNA but with no other DNAs tested. Genomic DNA pu rified from C. albicans blastoconidia suspended in blood was amplified by PCR with fungus-specific universal primers ITS3 and ITS4. With the C. albicans-specific probe labeled with digoxigenin, a biotinylated c apture probe, and streptavidin-coated microtitration plates, amplified DNA from as few as two C. albicans cells per 0.2 ml of blood could be detected by enzyme immunoassay.