Comparative use of quantitative PCR (qPCR), droplet digital PCR (ddPCR) and Recombinase Polymerase Amplification (RPA) in detection of Shiga Toxin-producing E. coli (STEC) in environmental samples

Authors: Ibekwe, Abasiofiok - Mark; MURINDA, SHLTON - California Polytechnic State University; Park, Stanley; OBAYIUWANA, AMARACHUKWU - Augustine University Ilara; MURRY, MARCIA - California Polytechnic State University; SHWARTZ, GREGORY - California Polytechnic State University; LUNDQUIST, TRYGVE - California Polytechnic State University

Submitted to: Water
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/10/2020
Publication Date: 12/13/2020
Citation: Ibekwe, A.M., Murinda, S.E., Park, S., Obayiuwana, A., Murry, M.A., Shwartz, G., Lundquist, T. 2020. Comparative use of quantitative PCR (qPCR), droplet digital PCR (ddPCR) and Recombinase Polymerase Amplification (RPA) in detection of Shiga Toxin-producing E. coli (STEC) in environmental samples. Water. 12(2). Article 3507. https://doi.org/10.3390/w12123507.
DOI: https://doi.org/10.3390/w12123507

Interpretive Summary: Shiga toxin producing E. coli, in particular serotype O157:H7 are prominent foodborne pathogens of public health and clinical significance. These pathogens are estimated to cause more than 265,000 illnesses in the United States, with more than 3,600 hospitalizations each year. In this study, we used recombinase polymerase amplification (RPA), real-time quantitative polymerase reaction, and droplet digital polymerase chain reaction assays for absolute and accurate quantification of E. coli O157:H7 from spiked and environmental samples. RPA assay detected Shiga toxin producing E. coli within 5-10 minutes at 39ºC with high sensitivity, and predictive value with limits of detection of about 5 to 50 colony forming units per milliliter of culture media. Real-time quantitative polymerase chain reaction assay exhibited higher quantification range of more than 1000 colony forming units per milliliter of culture media and droplet digital polymerase chain reaction had a quantification range from 10 to 1000 colony forming units per milliliter of culture media. The long term goal of these methods is to develop protocols that can be used directly in the field, at the point-of-care, for rapid real-time detection of pathogens followed by absolute quantification in the laboratory to confirm the prevalence of the pathogen in the field. This information will be of interest to produce growers, shippers, packers and processors, as well as livestock producers and food and feed processors and water utility agencies.

Technical Abstract: E. coli 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 E. coli O157:H7, real-time quantitative PCR (qPCR) for quantification, and droplet digital PCR (ddPCR) for absolute and accurate quantification of E. coli O157:H7 from spiked and environmental samples. Primer and probe sets were used for the detection of stx1 and stx2 using RPA. Genes encoding for stx1, stx2, eae, and rfbE were used to quantify E. coli O157:H7 in the water samples. Furthermore, duplex ddPCR assays were used to quantify the pathogens in these samples. Duplex assay set 1 used stx1 and rfbE genes, while assay set 2 used stx2 and eae genes. Droplet digital PCR was used for the absolute quantification of E. coli 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 104 CFU/mL with a high reproducibility. In addition, quantification by qPCR was from 103 to 107 CFU/mL of the wastewater samples. Therefore, the RPA assay has potential as a point of care tool for the detection of E. coli O157:H7 from different environmental sources, followed by quantification of the target concentrations.