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Applied and Environmental Microbiology, March 2003, p. 1775-1782, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1775-1782.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Evaluation of a Rapid, Quantitative Real-Time PCR Method for Enumeration of Pathogenic Candida Cells in Water

Nichole E. Brinkman,1 Richard A. Haugland,1* Larry J. Wymer,1 Muruleedhara Byappanahalli,2 Richard L. Whitman,2 and Stephen J. Vesper1

National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268,1 United States Geological Survey, Porter, Indiana 463042

Received 10 May 2002/ Accepted 11 December 2002

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5' nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

* Corresponding author. Mailing address: U. S. Environmental Protection Agency, 26 West M. L. King Dr., Mail Stop 314, Cincinnati, OH 45268. Phone: (513) 569-7135. Fax: (513) 569-7117. E-mail: haugland.rich{at}epa.gov.

Applied and Environmental Microbiology, March 2003, p. 1775-1782, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1775-1782.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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