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Quantification of microcystin‐producing cyanobacteria and E. coli in water by 5′‐nuclease PCR
Correspondence to: P.A. Horgen, Master of Biotechnology Program, Department of Botany, University of Toronto at Mississauga, 3359 Mississauga Rd. N, Mississauga, Ontario, L5L 1C6, Canada (e‐mail: phorgen@utm.utoronto.ca).
Abstract
Aims: 5′‐Nuclease (real‐time, quantitative) PCR methodologies were developed and applied as diagnostic tools for the detection of microcystin‐producing cyanobacteria and Escherichia coli in water.
Methods and Results: PCR was used to detect regions of the lacZ gene in E. coli, and the microcystin synthetase gene in microcystin‐producing cyanobacteria. In environmental water samples, natural inhibitors to PCR were effectively removed with a prefiltration step and an EDTA wash. A lower detection limit of 10 cells ml−1 was obtained with endpoint PCR detection. 5′‐Nuclease PCR was used for microbial quantification of 1 ml inoculated water samples. We were able to detect down to three copies of our target genes per sample within about 2 h (post‐DNA isolation) for both E. coli and microcystin‐producing cyanobacteria.
Conclusions: 5′‐Nuclease PCR offers a rapid and sensitive method of bacterial quantification in water samples.
Significance and Impact of Study: 5′‐Nuclease PCR can be adopted as an effective diagnostic tool for monitoring microbiological water quality, through coliform quantification, and detection of other waterborne microbial pathogens.
Citing Literature
Number of times cited: 29
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