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

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 as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157: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 of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C T ) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C T and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 ؋ 10 ؊5 pg of E. coli O157:H7 DNA ml ؊1 equivalent to approximately 6.4 ؋ 10 3 CFU of E. coli O157:H7 ml ؊1 based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were >3.5 ؋ 10 4 CFU g ؊1 . E. coli O157: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 ؊1 with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.Constructed wetlands are commonly used for biotreatment of urban runoff and agricultural waste, such as dairy waste wash water from milk cows and on-farm waste from manure piles. These wastes harbor different bacterial species including human pathogens. Present management practices for dairy wash water on most commercial farms involve long-term storage in ponds where evaporation, percolation into groundwater, or spraying onto crops and/or disposal on land occurs. A constructed wetland treatment system maximizes the utility of existing storage ponds, reduces sediment loading in the wash water ponds through on-site treatment, increases pond capacity, and decreases the need to clean and scrape storage ponds. The goal is to have a final effluent water from the wetland that is suitable for on-site reuse and reduces the amount of contaminants entering groundwater supplies as a result of percolation of wash water stored in ponds and sprayed on disposal lands. This final product is expected to enhance water and air quality; reduce biological oxygen demand, nitrate-nitrogen, phosphorus, and ammonia emissions; and inactivate protozoan parasites and pathogens present in dairy wash water.The wash waters from dairy and beef cattle operations harbor different bacterial species including human pathogens such as enterohe...

Evaluation of a Real-Time PCR Kit for Detecting Escherichia coli O157 in Bovine Fecal Samples

“…The detection range of 10 0 to 10 4 and minimum detection limit of Յ10 bacteria gram Ϫ1 of inoculated feces reported here for postenrichment samples are similar to other previously reported results (15,23). The assay detects all E. coli O157 isolates whether they have Shiga toxin or not.…”

Qualitative and quantitative methodologies for determination of airborne microorganisms at concentrated animal-feeding operations

“…in soil (Ibekwe and Grieve 2003;Ibekwe et al 2004), feces (Bono et al 2004), wash wastewater (Ibekwe et al 2002;Malorny et al 2007;Wolffs et al 2007), and food (Fortin et al 2001;Heller et al 2003;Ellingson et al 2004). While the detection limit for these pathogens is generally [10 2 c.f.u.…”

“…The number of copies of a target gene in an aerosol or other environmental sample (e.g., soil, water, food) is determined by monitoring the increase in the amplicon concentration during PCR and then regressing to the original concentration. Standard curves can be prepared by serially diluting genomic DNA that has been isolated from a pure bacterial culture (Ibekwe et al 2002;Peccia and Hernandez 2006). A relationship between the quantity of DNA and colony forming units (c.f.u.)…”

Using a real-time quantitative polymerase chain reaction (PCR) method for reliable enumeration of Aeromonas hydrophila in water samples

“…The slopes of standard curves were -3·33, -3·47 and -3·34 for 16SrRNA gene, aerA and ast respectively (Figure 3). The standard curves showed a linear correlation between Ct values and cell numbers ranging from 10 8 to 10 1 cells; the amplification efficiency was 99.7% for 16SrRNA, 99.3% for aerA and 94.2% for ast genes (calculated according to (Ibekwe et al, 2002)). The results show that the quantification limit of the SYBR Green assay was 10 cells.…”

“…The lower limit of quantification for the assay was set to be 1 pg per reaction. The slopes obtained were -3.29, -3.21 and -3.43 for 16SrRNA, aerA and ast respectively and conesquently, very high efficiencies (> 95%) were obtained after calculation according to (Ibekwe et al, 2002). Based on the standard curves and the limit of detection of this assay, negative results were defined as those exhibiting CT values higher than 35.…”