Multiplex Fluorogenic Real-Time PCR for Detection and Quantification ofEscherichia coliO157:H7 in Dairy Wastewater Wetlands

Open Access
Publisher Website
Google Scholar
Abstract
Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such asEscherichia colifrom urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantifyE. coliO157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae)gene ofE. coliO157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33E. coliO157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (CT) and the starting quantity ofE. coliO157:H7 DNA. A similar correlation was observed between theCTand number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10−5pg ofE. coliO157:H7 DNA ml−1equivalent to approximately 6.4 × 103CFU ofE. coliO157:H7 ml−1based on plate counts was determined. Quantification ofE. coliO157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 104CFU g−1.E. coliO157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g−1with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination ofE. coliO157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.