| Summary: | <i>E. coli</i> O157:H7 is a foodborne pathogen that constitutes a global threat to human health. However, the quantification of this pathogen in food and environmental samples may be problematic at the low cell numbers commonly encountered in environmental samples. In this study, we used recombinase polymerase amplification (RPA) for the detection of <i>E. coli</i> O157:H7, real-time quantitative PCR (qPCR) for quantification, and droplet digital PCR (ddPCR) for absolute and accurate quantification of <i>E. coli</i> O157:H7 from spiked and environmental samples. Primer and probe sets were used for the detection of <i>stx</i>1 and <i>stx</i>2 using RPA. Genes encoding for <i>stx</i>1, <i>stx</i>2, <i>eae</i>, and <i>rfb</i>E were used to quantify <i>E. coli</i> O157:H7 in the water samples. Furthermore, duplex ddPCR assays were used to quantify the pathogens in these samples. Duplex assay set 1 used <i>stx</i>1 and <i>rfb</i>E genes, while assay set 2 used <i>stx</i>2 and <i>eae</i> genes. Droplet digital PCR was used for the absolute quantification of <i>E. coli</i> O15:H7 in comparison with qPCR for the spiked and environmental samples. The RPA results were compared to those from qPCR and ddPCR in order to assess the efficiency of the RPA compared with the PCR methods. The assays were further applied to the dairy lagoon effluent (DLE) and the high rate algae pond (HRAP) effluent, which were fed with diluted DLE. The RPA detected was <10 CFU/mL, while ddPCR showed quantification from 1 to 10<sup>4</sup> CFU/mL with a high reproducibility. In addition, quantification by qPCR was from 10<sup>3</sup> to 10<sup>7</sup> CFU/mL of the wastewater samples. Therefore, the RPA assay has potential as a point of care tool for the detection of <i>E. coli</i> O157:H7 from different environmental sources, followed by quantification of the target concentrations.
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