Development of a rapid quantitative PCR assay for direct detection and quantification of culturable and non-culturable Escherichia coli from agriculture watersheds |
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Authors: | Khan Izhar U H Gannon Vic Kent Rob Koning Wendell Lapen David R Miller Jim Neumann Norman Phillips Rob Robertson Will Topp Edward van Bochove Eric Edge Thomas A |
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Institution: | National Water Research Institute, Aquatic Ecosystem Protection Research Division, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, Canada. |
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Abstract: | A real-time quantitative polymerase chain reaction (Q-PCR) assay was developed for detecting and quantifying Escherichia coli in water samples from agricultural watersheds. The assay included optimization of DNA extraction and purification from water samples, and Q-PCR amplification conditions using newly designed species-specific oligonucleotide primers derived from conserved flanking regions of the 16S rRNA gene, the internal transcribed spacer region (ITS) and the 23S rRNA gene. The assay was optimized using a pure culture of E. coli with known quantities spiked into autoclaved agricultural water samples. The optimized assay was capable of a minimum quantification limit of 10 cells/ml of E. coli in the spiked agricultural water samples. A total of 121 surface water samples from three agricultural watersheds across Canada were analyzed, and results were compared with conventional culture-based enumerations of E. coli. The Q-PCR assay revealed significantly higher numbers of E. coli in water samples than the culture-based assay in each agricultural watershed. The new Q-PCR assay can facilitate the quantification of E. coli in a single water sample in < 3 h, including melt curve analysis, across a range of agricultural water quality conditions. |
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