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Title: Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification

Author
item MURINDA, SHELTON - California Polytechnic State University
item Ibekwe, Abasiofiok - Mark
item ZULKAFFLY, SYAIZUL - California Polytechnic State University
item CRUZ, ANDREW - California Polytechnic State University
item PARK, STANLEY - California Polytechnic State University
item RAZAK, NUR - California Polytechnic State University
item PAUDZAI, FARAH - California Polytechnic State University
item AB SAMAD, LIANA - California Polytechnic State University
item BAQUIR, KHAIRUL - California Polytechnic State University
item MUTHAIYAH, KOKILAH - California Polytechnic State University
item SANTIAGO, BRENNA - California Polytechnic State University
item RUSLI, AMIRUL - California Polytechnic State University
item BALKCOM, SEAN - California Polytechnic State University

Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/21/2014
Publication Date: 4/21/2014
Citation: Murinda, S.E., Ibekwe, A.M., Zulkaffly, S., Cruz, A., Park, S., Razak, N., Paudzai, F.M., Ab Samad, L., Baquir, K., Muthaiyah, K., Santiago, B., Rusli, A., Balkcom, S. 2014. Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification. Foodborne Pathogens and Disease. 11(7):529-536.

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 and 30 deaths each year. In this study, we focused on designing and evaluating recombinase polymerase amplification (RPA) primers and fluorescent probes that were used for isothermal detection of Shiga toxin producing E. coli in real-time. The 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. The long term goal of this method will be to develop protocols that can be used directly in the field, at the point-of-care, for rapid real-time detection of pathogens. 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: Shiga toxin (Stx) producing E. coli (STEC) are a major family of foodborne pathogens of immense public health, zoonotic and economic significance in the US and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal of this study was to assess the potential application of RPA in detection of Shiga toxin producing E. coli. This study focused on designing and evaluating RPA primers and fluorescent probes for isothermal (39ºC) detection of STEC. Compatible sets of candidate primers and probes were designed for detection of Stx1 and 2, respectively, and were evaluated for specificity and sensitivity against STEC (n=12) of various stx genotypes (stx1/stx2, stx1 or stx2, respectively), including non-Stx-producing E. coli (n=28) and other genera (n=7). The primers and probes that were designed targeted amplification of the subunit A moiety of stx1 and stx2. The assay detected STEC in real-time (within 5-10 minutes at 39ºC) with high sensitivity (93.5% vs. 90%; stx1 vs. stx2), specificity (99.1% vs. 100%; stx1 vs. stx2) and predictive value (97.9% for both stx1 vs. stx2). Limits of detection of ~5 to 50 CFU/ml were achieved in serially diluted cultures grown in Brain Heart Infusion broth. This study successfully demonstrated for the first time that RPA can be used for isothermal real-time detection of STEC.