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

Abstract

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.Yeasts are a significant component of the microbiota of most natural aquatic ecosystems (17, 33) and can also occur in drinking water distribution systems as a result of their ability to survive treatment practices and become incorporated into biofilms (6,12,22,30,31). The majority of these organisms have no known human health effect. However, a small number of species, primarily within the anamorphic genus Candida, are important opportunistic pathogens (23).The importance of pathogenic Candida as agents of nosocomial infections has led to the development of a number of modern molecular diagnostic methods to facilitate their detection and identification in clinical samples. Methods based on the PCR and DNA hybridization probes have received particular attention (9,25,26,32,39). The more recent advent of fluorescent probe-based PCR technology (21) has led to the development of homogeneous methods for detecting these organisms that require relatively short periods of time to perform (16,28).Quantitative PCR (QPCR) has been demonstrated to be useful for quantitative analysis of microorganisms in environmental samples (29,34,35,36), but, to our knowledge, this approach has not been used in the analysis of yeasts in water. Analyses for pathogenic yeasts in drinking or recreational water systems have the potential to expedite the identification of possible health hazards resulting either directly from the presence of these organisms or, as their presence might indicate, indirectly from other waterborne pathogens.The first object...

Development of real-time PCR using Minor Groove Binding probe to monitor the biological control agent Candida oleophila (strain O)

“…On the other hand, fair differences were observed between the average population densities obtained by real-time PCR and those obtained by plating on semi-selective media which has been already used to monitor strain O population on apple surface (De Clercq et al, 2001). Such similarities were also obtained by Brinkman et al (2003) with some Candida species. Nevertheless, the population densities obtained by both methods are difficult to compare.…”

“…The competitive PCR has important drawbacks (time consuming, laborious protocol, cross-contamination of PCR products) and has been overtaken by the development of real-time PCR. Real-time PCR using the Taqman fluorescent chemistry has already been used to quantify microorganisms in their environment, like strains of Candida species in water samples (Brinkman et al, 2003), Stachybotrys chartarum in water and dust (Haugland et al, 2002) or plant pathogens in soil (Filion et al, 2003b). A more sophisticated Taqman probe, called 3V-Minor Groove Binder-DNA (MGB) probes, can be used to modify the detection specificity (Kutyavin et al, 2000;Marbot et al, 2003;Salmon, 2002).…”

Comparison of mold concentrations quantified by MSQPCR in indoor and outdoor air sampled simultaneously

“…Methods have been reported previously for extracting DNA and performing MSQPCR analyses (Haugland et al, 2002;Brinkman et al, 2003;Haugland et al, 2004). All primer and probe sequences, as well as known species comprising the assay groups, were published at the website: http://www.epa.gov/microbes/moldtech.htm.…”

Relative moldiness index as predictor of childhood respiratory illness

“…Methods have been reported previously for preparing conidial suspensions from fungal cultures, extracting DNA, performing MSQPCR analyses, and preparing standard calibration curves for target conidia versus delta cycle threshold values (ΔC T = C T,target − C T,reference ), using co-extracted DNA from Geotrichum candidum as an exogenous reference (Haugland et al, 2002;Brinkman et al, 2003;Haugland et al, 2004). All primer and probe sequences, as well as known species comprising the assay groups, were published at the website: http://www.epa.gov/microbes/moldtech.htm.…”