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Title: MULTIPLEX FLUOROGENIC REAL-TIME (TAQMAN) PCR FOR DETECTION AND QUANTIFICATION OF ESCHERICHIA COLI O157:H7 IN DAIRY WASTEWATER WETLANDS

Author
item Ibekwe, Abasiofiok - Mark
item Watt, Pamela
item Grieve, Catherine
item Sharma, Vijay
item LYONS, STEVEN - OC WATER DISTRICT, CA

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/17/2002
Publication Date: N/A
Citation: N/A

Interpretive Summary: E. coli serotype O157:H7 strains have been particularly important as serious human pathogens in recent years and cattle are important reservoirs. These bacteria can be transmitted on contaminated food products, including produce consumed fresh or subjected to mild processing. In this study, DNA was used to detect and quantify E. coli O157:H7 in dairy wastewater prior to and following treatment by the wetlands. The objective of this study was to develop a real-time multiplex PCR assay and explore the potential of using this assay to estimate cell numbers of E. coli O157:H7 in environmental samples. An on-farm model was used to determine the effectiveness of a sub-surface constructed wetland system in reducing E. coli O157:H7 from dairy waste washwater. Agricultural wastes must be treated prior to disposal and in this study we found that constructed wetlands in combination with stabilization pond is a good potential biological treatment option to agricultural waste.

Technical Abstract: Food and water-borne illnesses are increasingly linked to intensive agricultural activities, and wastewater must first be treated for the removal of different compounds and pathogenic bacteria before land application. In this study, a multiplex fluorogenic PCR assay was used to quantity Escherichia coli O157:H7 in soil, manure, feces, and dairy wastewater. Primers and probes were designed to amplify and quantify stx1, stx2, and eaeA genes of Escherichia coli O157:H7 in one reaction. Primer specificity was confirmed with DNA from 9 Escherichia coli O157:H7 and related strains with and without the three genes. The linearity between the threshold fluorescence value and the quantity of E. coli O157:H7 DNA was measured and relate to colony forming units of the pure culture. A detection limit of 6400 E. coli O157:H7 based on plate counts was found. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were about 35000.These results showed that quantitative determination of E. coli O157:H7 in the environment is possible, and represents a considerable advancement in pathogen quantification in different ecosystems.