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Journal of Clinical Microbiology, January 1999, p. 165-170, Vol. 37, No. 1
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Rapid Identification of up to Three Candida Species in a Single Reaction Tube by a 5' Exonuclease Assay Using Fluorescent DNA Probes

Jong Hee Shin,1 Frederick S. Nolte,2 Brian P. Holloway,3 and Christine J. Morrison4,*

Department of Clinical Pathology, Chonnam University Medical School, Kwangju, Korea,1 and Department of Clinical Laboratory Medicine, Emory University Hospital,2 and Scientific Resources Program3 and Division of Bacterial and Mycotic Diseases,4 National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

Received 3 August 1998/Returned for modification 3 September 1998/Accepted 15 October 1998

We used fungus-specific PCR primers and species-specific DNA probes to detect up to three Candida species in a single reaction tube by exploiting the 5' to 3' exonuclease activity of Taq 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), tetrachloro-6-carboxy-fluorescein (TET), or hexachloro-6-carboxy-fluorescein (HEX), and at the 3' end with a quencher dye, 6-carboxy-tetramethyl-rhodamine. During PCR amplification, each reporter dye emits a characteristic wavelength 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-FAM, 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. CG-FAM and CK-TET were added to a second tube to simultaneously detect the typically more innately fluconazole-resistant species C. glabrata and C. krusei, respectively. All-CAN-TET, a Candida genus probe, was added to a third tube to detect DNAs from all Candida species tested. 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 for mixed fungemias, were tested. Control samples included those from blood culture bottles with no growth (n = 10) or from patients with confirmed bacteremia (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 incubation steps. It is sensitive and specific for the detection and identification of Candida species from blood culture bottles, including those containing mixtures of Candida species, and should facilitate an earlier specific diagnosis, leading to more appropriately targeted antifungal drug therapy.

* Corresponding author. Mailing address: Mailstop G-11, CDC, 1600 Clifton Rd., N.E., Atlanta, GA 30333. Phone: (404) 639-3098. Fax: (404) 639-3546. E-mail: cjm3{at}cdc.gov.

Journal of Clinical Microbiology, January 1999, p. 165-170, Vol. 37, No. 1
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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