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Article

Comparative Use of Quantitative PCR (qPCR), Droplet Digital PCR (ddPCR), and Recombinase Polymerase Amplification (RPA) in the Detection of Shiga Toxin-Producing E. coli (STEC) in Environmental Samples

1
US Salinity Laboratory, USDA-ARS, 450 W. Big Springs Rd., Riverside, CA 92507, USA
2
Animal and Veterinary Sciences Department, California State Polytechnic University, Pomona, CA 91768, USA
3
Department of Biological Sciences, Augustine University Ilara-Epe, Lagos State 106101, Nigeria
4
Biological Sciences Department, California State Polytechnic University, Pomona, CA 91768, USA
5
BioResource and Agricultural Engineering Department, College of Agriculture, California Polytechnic State University, San Luis Obispo, CA 91768, USA
6
Civil and Environmental Engineering Department, College of Engineering, California Polytechnic State University, San Luis Obispo, CA 91768, USA
*
Author to whom correspondence should be addressed.
Water 2020, 12(12), 3507; https://doi.org/10.3390/w12123507
Received: 5 October 2020 / Revised: 4 December 2020 / Accepted: 10 December 2020 / Published: 13 December 2020
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. View Full-Text
Keywords: Shiga toxin-producing E. coli; high rate algae ponds; dairy lagoon effluent; quantitative PCR; droplet digital PCR; recombinase polymerase amplification Shiga toxin-producing E. coli; high rate algae ponds; dairy lagoon effluent; quantitative PCR; droplet digital PCR; recombinase polymerase amplification
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MDPI and ACS Style

Ibekwe, M.A.; Murinda, S.E.; Park, S.; Obayiuwana, A.; Murry, M.A.; Schwartz, G.; Lundquist, T. Comparative Use of Quantitative PCR (qPCR), Droplet Digital PCR (ddPCR), and Recombinase Polymerase Amplification (RPA) in the Detection of Shiga Toxin-Producing E. coli (STEC) in Environmental Samples. Water 2020, 12, 3507. https://doi.org/10.3390/w12123507

AMA Style

Ibekwe MA, Murinda SE, Park S, Obayiuwana A, Murry MA, Schwartz G, Lundquist T. Comparative Use of Quantitative PCR (qPCR), Droplet Digital PCR (ddPCR), and Recombinase Polymerase Amplification (RPA) in the Detection of Shiga Toxin-Producing E. coli (STEC) in Environmental Samples. Water. 2020; 12(12):3507. https://doi.org/10.3390/w12123507

Chicago/Turabian Style

Ibekwe, Mark A.; Murinda, Shelton E.; Park, Stanley; Obayiuwana, Amarachukwu; Murry, Marcia A.; Schwartz, Gregory; Lundquist, Trygve. 2020. "Comparative Use of Quantitative PCR (qPCR), Droplet Digital PCR (ddPCR), and Recombinase Polymerase Amplification (RPA) in the Detection of Shiga Toxin-Producing E. coli (STEC) in Environmental Samples" Water 12, no. 12: 3507. https://doi.org/10.3390/w12123507

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