Evaluation of a real-time Taqman® PCR method for assessment of pathogenic coliform contamination in sediment: A risk-based approach

Autor: V. R. Hinton, Herbert L. Fredrickson, J. S. Furey, K. Betts, Karl J. Indest
Rok vydání: 2004
Předmět:
Zdroj: Aquatic Ecosystem Health & Management. 7:415-424
ISSN: 1539-4077
1463-4988
Popis: Management of contaminated sediments has focused predominately on chemical agents, overshadowing risks posed by pathogenic microorganisms. Current accepted bacterial indicator methods do not provide defensible data with respect to the occurrence and types of pathogens in sediments. In an effort to adapt new defensible methods for assessing the risk posed by pathogens in sediments, we evaluated the sensitivity of a commercially available real-time polymerase chain reaction TaqMan® Escherichia coli 0157:H7detection kit. The lower limit of linear quantitation of this assay was experimentally determined in sediment and sediment extract samples spiked with known amounts of E. coli 0157:H7 DNA. Parallel control experiments were conducted in pure water samples spiked with known amounts of Escherichia coli 0157:H7 DNA. The lower limit of quantification of the TaqMan® assay was 1000 colony forming units when interrogating 100 mg sediment samples. In contrast, the assay was 20-fold more sensitive with a lower limit of quantification of 50 colony forming units in pure water and sediment extract samples. These results suggest that the sensitivity of the TaqMan® Escherichia coli 0157:H7detection kit is more dependent on recovery of the desired target from the sediment matrix than efficiency of polymerase chain reaction amplification. The potential human health risk associated with the lower limit of quantification of the TaqMan® assay in the spiked sediment samples was estimated using a Beta-Poisson dose-response model. Using this approach, lower limit values corresponded to exposure levels of Escherichia coli 0157:H7 that meet United States Environmental Protection Agency accepted illness rates for recreational swimming.
Databáze: OpenAIRE
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