Quantification of microcystin-producing cyanobacteria and E. coli in water by 5'-nuclease PCR.
OBJECTIVE
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.


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.


CONCLUSIONS
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.
Foulds
I V
IV
Department of Botany, University of Toronto at Mississauga, Mississauga, Ontario, Canada.
Granacki
A
A
Xiao
C
C
Krull
U J
UJ
Castle
A
A
Horgen
P A
PA
D000458:Cyanobacteria / Q000302:isolation & purification* / Q000378:metabolism; D004247:DNA / Q000032:analysis / Q000302:isolation & purification; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005618:Fresh Water / Q000737:chemistry; D052998:Microcystins; D010456:Peptides, Cyclic / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014871:Water Microbiology
D017931:DNA Primers; D052998:Microcystins; D010456:Peptides, Cyclic; C078588:microcystin; D004247:DNA