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Journal of Clinical Microbiology, May 2004, p. 2085-2093, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.2085-2093.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Rapid Quantification of Drug Resistance Gene Expression in Candida albicans by Reverse Transcriptase LightCycler PCR and Fluorescent Probe Hybridization

Joao P. Frade,1,2 David W. Warnock,2 and Beth A. Arthington-Skaggs2*

Department of Microbiology, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal,1 Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 303332

Received 11 November 2003/ Returned for modification 8 January 2004/ Accepted 26 January 2004

We developed a rapid, sensitive, and reproducible assay to quantify Candida albicans ACT1, CDR1, CDR2, ERG11, and MDR1 mRNA using a two-step reverse transcription and LightCycler real-time PCR (RT-LightCycler PCR) method with sequence-specific hybridization probes. We compared RT-LightCycler PCR with Northern hybridization for quantitative analysis of gene expression in isolates with various fluconazole susceptibilities. Specificity of each LightCycler PCR was verified by LightCycler melting curve analysis and agarose gel electrophoresis of amplified products. Correlation of quantification results between RT-LightCycler PCR and Northern hybridization yielded correlation coefficients of >=0.91 for all genes except MDR1 (0.74). In this case, reduced correlation was due to the inability of Northern hybridization to accurately quantify the high MDR1 expression in a susceptible dose-dependent isolate which was shown by RT-LightCycler PCR to overexpress MDR1 >200-fold relative to the other isolates tested. In four isolates, low levels of CDR2 mRNA were detected by RT-LightCycler PCR but were undetectable by Northern hybridization. mRNA quantification by RT-LightCycler PCR correlates with Northern hybridization and offers additional advantages, including increased sensitivity and speed of analysis, along with lower RNA concentration requirements and an increased dynamic range of signal detection.

* Corresponding author. Mailing address: Mycotic Diseases Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd., N.E., Mailstop G-11, Atlanta, GA 30333. Phone: (404) 639-4041. Fax: (404) 639-3546. E-mail: bskaggs{at}cdc.gov.

Journal of Clinical Microbiology, May 2004, p. 2085-2093, Vol. 42, No. 5
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.5.2085-2093.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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