A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples

Behets, Jonas; Declerck, Priscilla; Delaedt, Yasmine; Verelst, Lieve; Ollevier, Frans

doi:10.1016/j.watres.2006.10.003

Cited by 48 publications

(33 citation statements)

References 33 publications

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“…Before we applied realtime PCR detection, the sensitivity of the assays was rigorously evaluated by amplifying known concentrations of DNA from target pathogens. The lower limits of detection ranged from 5 to 10 copies ( Table 2), indicating that the detection sensitivity values of our real-time PCR assay were comparable to the values reported in the literature (3,31). The cross-reactivity of each primer set for each target has also been assed by testing a number of other microorganisms, including B. vulgatus ATCC 8482, A. hydrophila ATCC 7966, C. coli ATCC 43478, L. pneumophila ATCC 33152, and Salmonella serovar Typhimurium ATCC 14028.…”

Section: Discussionsupporting

confidence: 83%

Real-Time PCR Detection of Pathogenic Microorganisms in Roof-Harvested Rainwater in Southeast Queensland, Australia

Ahmed

Huygens

Goonetilleke

et al. 2008

Appl Environ Microbiol

162

138

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In this study, the microbiological quality of roof-harvested rainwater was assessed by monitoring the concentrations of Escherichia coli, enterococci, Clostridium perfringens, and Bacteroides spp. in rainwater obtained from tanks in Southeast Queensland, Australia. Samples were also tested using real-time PCR (with SYBR Green I dye) for the presence of potential pathogenic microorganisms. Of the 27 rainwater samples tested, 17 (63%), 21 (78%), 13 (48%), and 24 (89%) were positive for E. coli, enterococci, C. perfringens, and Bacteroides spp., respectively. Of the 27 samples, 11 (41%), 7 (26%), 4 (15%), 3 (11%), and 1 (4%) were PCR positive for the Campylobacter coli ceuE gene, the Legionella pneumophila mip gene, the Aeromonas hydrophila lip gene, the Salmonella invA gene, and the Campylobacter jejuni mapA gene. Of the 21 samples tested, 4 (19%) were positive for the Giardia lamblia ␤-giardin gene. The binary logistic regression model indicated a positive correlation (P < 0.02) between the presence/absence of enterococci and A. hydrophila. In contrast, the presence/ absence of the remaining potential pathogens did not correlate with traditional fecal indicators. The poor correlation between fecal indicators and potential pathogens suggested that fecal indicators may not be adequate to assess the microbiological quality of rainwater and consequent health risk.

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Section: Discussionsupporting

confidence: 83%

Real-Time PCR Detection of Pathogenic Microorganisms in Roof-Harvested Rainwater in Southeast Queensland, Australia

Ahmed

Huygens

Goonetilleke

et al. 2008

Appl Environ Microbiol

162

138

View full text Add to dashboard Cite

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“…This inhibition is due to competition between the internal control with the target, when this target is present in excess. Comparable results have been described by the IPC manufacturer and in the literature (Behets et al, 2007;Hallett and Bartholomew, 2009;McBeath et al, 2006). Similar values have been found by performing the same spiking experiment using a singleplex approach (i.e.…”

Section: Determination Of the Taqman Assay Sensitivitysupporting

confidence: 87%

Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix

Vanysacker

Denis

Roels

et al. 2014

Journal of Microbiological Methods

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Candidatus Microtrhix parvicella is one of the most common filamentous bacteria reported to be involved in bulking and foaming problems in activated sludge plants worldwide. In order to detect and quantify both M. parvicella and Microthrix calida by quantitative PCR (qPCR), primers targeting 16S rDNA genes were designed. The qPCR reaction was optimized by using the TaqMan technology and an internal positive control was included to ensure the absence of PCR inhibitors. A total of 29 samples originating from different wastewater treatment plants were analyzed and the results were compared by using conventional microscopy, fluorescent in situ hybridization and an existing SYBR Green-based assay. Our assay showed a 100% specificity for both M. parvicella and M. calida, a sensitivity of 2.93 × 10 9 to 29 copy numbers/reaction, an amplification efficiency of 93% and no PCR inhibition. By performing a spiking experiment including different Microthrix concentrations, recovery rates ranging from 65 to 98% were obtained. A positive correlation with the SYBR Green assay (R 2 = 0.85) was found and most of the samples were in accordance with the microscopical observation. In comparison with SYBR Green assay, the probe-based TaqMan assay had a much lower detection limit. Compared with microscopy, some samples had a lower or higher enumeration when using qPCR. In conclusion, a qPCR method is forwarded here that could be useful as an early warning tool for fast and reliable detection of Microthrix in for instance sludge bulking events.

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“…The qPCR detection limit was found to be 10 fg for E. coli, which corresponds to ϳ2 copies of the uidA gene (40,43). The same detection limits, which are lower than the limits reported previously (7,41), were also found for the P. aeruginosa, K. pneumoniae, and A. hydrophila target genes regA, phoE, and aha1, respectively. The efficiency of qPCR was found to be 94.9% for E. coli.…”

Section: Methodssupporting

confidence: 72%

Influence of Air Quality on the Composition of Microbial Pathogens in Fresh Rainwater

Kaushik

Balasubramanian

Cruz

2012

Appl Environ Microbiol

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In this study, the microbiological quality of fresh rainwater was assessed from 50 rain events under tropical weather conditions for a year. The levels of four major opportunistic waterborne pathogens, namely, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Aeromonas hydrophila, in rainwater samples were quantified by using a robust and sensitive quantitative PCR (qPCR) method. Of the 50 rainwater samples, 25 were found to be positive for at least one pathogen: 21 for E. coli, 16 for P. aeruginosa, 6 for K. pneumoniae, and 1 for A. hydrophila. In addition to the microbiological assessment of rainwater samples, we also studied the influence of prevailing air quality on the microbial quality of rainwater over the sampling period. A significant change in the diversity and relative abundance of the basic microbial indicator organisms in rainwater was observed during a major regional air pollution episode in Southeast Asia due to biomass-burning emissions. Water shortage is becoming a major problem worldwide due to the increased demands of growing population and urbanization (29,38). In an effort to ensure a sustainable future, a global search for alternative sources of water is ongoing, and rainwater harvesting has received considerable attention (1,13,23,44,45). However, the use of rainwater for drinking purposes is impeded by issues of water quality in terms of chemical and microbiological contamination and its potential health risks (1). While numerous studies on the chemical contamination of rainwater have been conducted (5,11,27,30,43), its microbial quality remains relatively unknown due to the analytical challenge involved in the detection of a range of contaminants, particularly pathogens.Some previous studies have reported that roof-harvested rainwater is generally acceptable for drinking and household purposes (12,13,16,26), while more-recent studies have revealed the presence of waterborne pathogens in rainwater samples (1,3,11,22,34,35,43). Differences in the characteristics of the catchment area, the type of water tanks, their cleanliness, and other factors, such as roof material, the intensity of human activities in the catchment area (urban or rural), and the presence of insects and birds on the rooftop, could be responsible for the contradictory results in the literature on the microbial quality of rainwater (10,17,18,34,39,46). Numerous studies conducted on the chemical composition of rainwater and roof runoff have demonstrated links between chemical contaminants and weather patterns, and between atmospheric transport and deposition (9,19,24,48). However, little is known about such links with respect to microbial composition. In order to identify sources of microorganisms in roof-harvested rainwater, it is desirable to study the microbial flora of fresh rainwater prior to its collection and storage, with no contamination from external sources.Microbial water quality monitoring is generally directed toward indicator microorganisms such as coliforms, Escherichia coli, and Klebsiel...

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A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples

Cited by 48 publications

References 33 publications

Real-Time PCR Detection of Pathogenic Microorganisms in Roof-Harvested Rainwater in Southeast Queensland, Australia

Real-Time PCR Detection of Pathogenic Microorganisms in Roof-Harvested Rainwater in Southeast Queensland, Australia

Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix

Influence of Air Quality on the Composition of Microbial Pathogens in Fresh Rainwater

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