Development of a rapid quantitative PCR assay for direct detection and quantification of culturable and non-culturable Escherichia coli from agriculture watersheds.
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.
Khan
Izhar U H
IU
National Water Research Institute, Aquatic Ecosystem Protection Research Division, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, Canada.
Gannon
Vic
V
Kent
Rob
R
Koning
Wendell
W
Lapen
David R
DR
Miller
Jim
J
Neumann
Norman
N
Phillips
Rob
R
Robertson
Will
W
Topp
Edward
E
van Bochove
Eric
E
Edge
Thomas A
TA
D000383:Agriculture; D000818:Animals; D002415:Cats; D002417:Cattle; D002648:Child; D015169:Colony Count, Microbial; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D021903:DNA, Ribosomal Spacer / Q000032:analysis; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D005618:Fresh Water / Q000382:microbiology*; D006168:Guinea Pigs; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D017422:Sequence Analysis, DNA; D013997:Time Factors
D003470:Culture Media; D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S