Rapid Detection and Absolute Quantification of E. coli O157:H7 in Environmental Samples Using Recombinase Polymerase Amplification (RPA) Followed By Droplet Digital PCR.

Fast and reliable detection and absolute quantification of E. coli O157:H7 in food and environmental sample without enrichment is a critical factor for food safety and public health. However, enumeration of this pathogen in food and environmental samples is not always accurate and quantitative. In this study, we used recombinase polymerase amplification (RPA) for the rapid detection of the pathogen, followed by real time quantitative PCR (qPCR) for quantification and droplet digital polymerase chain reaction (ddPCR) for absolute and accurate quantification without external standards. Using the stx1, stx2, eae, and the rfbE genes of E. coli O157:H7, we evaluated the specificity and detection limit of E. coli O157:H7 in soil and water samples inoculated with the pathogen. The assay was further applied to swine, dairy, beef, and duck manure, and waste water effluent collected from a dairy wetland over a twelve-month period. RPA was used to assess detection limit of this pathogen in comparison to enrichment, while ddPCR was used to determine the absolute quantification of the pathogen in relation to qPCR for samples derived from heterogeneous matrices that often include inhibitors and a non-target bacterial background flora. ddPCR showed a high degree of linearity and quantification in a dynamic range from 1 to 10⁴ CFU/mL/g with high reproducibility. qPCR exhibited a dynamic range from 10³ to 10⁷ CFU/mL/g of waste water/soil sample. Quantification of environmental samples by ddPCR provided 75% sensitivity and 80% specificity in distinguishing E. coli O157:H7 from environmental samples.