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Quantification of microcystin-producing cyanobacteria and E. coli in water by 5'-nuclease PCR
I V Foulds1, A Granacki, C Xiao
1Department of Botany, University of Toronto at Mississauga, Mississauga, Ontario, Canada.
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 The 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.
