Rapid identification of up to three Candida species in a single reaction tube by a 5 ' exonuclease assay using fluorescent DNA probes

We used fungus-specific PCR primers and species-specific DNA probes to dete ct up to three Candida species in a single reaction tube by exploiting the 5' to 3' exonuclease activity of Tag DNA polymerase. Probes to the internal transcribed spacer region of the rRNA gene were labeled at the 5' end with one of three fluorescent reporter dyes, 6-carboxy-fluorescein (FAM), tetra chloro-6-carboxy-fluorescein (TET), or hexachloro-6-carboxy-fluorescein (HE X), and at the 3' end with a quencher dye, 6-carboxy-tetramethyl-rhodamine. During PCR amplification, each reporter dye emits a characteristic wavelen gth as it is cleaved from its specific target DNA and from the quencher dye . Therefore, signals from up to three probes can be detected simultaneously during the PCR assay. Six probes were designed for use in this study: CA-F AM, CT TET, and CP-HEX were added to one tube to simultaneously detect the typically fluconazole-sensitive species C. albicans, C. tropicalis, and C. parapsilosis, respectively. CC-PAM and CK-TET were added to a second tube t o simultaneously detect the typically more innately fluconazole-resistant s pecies C. glabrata and C. krusei, respectively. AII-CAN-TET, a Candida genu s probe,pas added to a third tube to detect DNAs from all Candida species t ested. DNAs recovered from 61 blood culture bottles, including 23 positive for C. albicans, 18 positive for C. glabrata, 6 positive for C. tropicalis, 6 positive for C. krusei, 5 positive for C. parapsilosis, and 3 positive f or mixed fungemias, were tested. Control samples included those from blood culture bottles with no growth (n = 10) or from patients with confirmed bac teremia (n = 10). Probes detected and correctly identified the organisms in 58 of 61 specimens (95.1%) and gave no false-positive results. This method is simple and rapid and does not require post-PCR hybridization and incuba tion steps. It is sensitive and specific for the detection and identificati on of Candida species from blood culture bottles, including those containin g mixtures of Candida species, and should facilitate an earlier specific di agnosis, leading to more appropriately targeted antifungal drug therapy.