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PROJECT SUMMARYCost-effective on-site assay technologies for pathogen detection are sorely lacking, even though they areessential in solving many global challenges such as food and water borne infections and disease outbreaks.For example, each year foodborne diseases from E. coli and salmonella contamination alone cost $6.4 billionin the US. One of the most common strategies for foodborne pathogen detection is laboratory-basedpolymerase chain reaction (PCR) of a single pathogen type that takes about 24 h followed by confirmationusing culture-based methods that take a minimum of 1 week. Additionally, a majority of techniques reli only onDNA whereas RNA is a better indicator of viable bacteria. Ideal technology should target detection of multiplepathogens through the detection of specific nucleic acid in a portable on-site detection platform. However, thedetection of multiple nucleic acid targets in a single assay is still problematic, and transitioning a robust,multiplexed nucleic acid detection assay into a low-cost, rapid, on-site detection device is a significantchallenge. To that end, our overall goal is to develop a flow-strip multiplex pathogen detection platform that willfulfill the following criteria: simple sample preparation, rapid multiplexed DNA/RNA target amplification andincorporation of reporter in a single pot, portable platform with colorimetric detection for on-site andinexpensive monitoring. Here, we propose to use recombinase polymerase amplification (RPA) and reversetranscriptase-RPA to incorporate unique zinc-finger protein (ZFP) binding motifs (referred to as ?Ztags?) andreporter moieties into the amplification product from nucleic acid targets of interest in order to detect thepresence of viable pathogen. The amplified product will be captured using immobilized ZFPs via specific ZFP-Ztag interaction for real-time reporter-based detection on a flow-strip platform. Our preliminary datademonstrated that we can incorporate both Ztag and reporter into the target nucleic acid using the RPAmethod and detect as low as 10 copies of target. Additionally, the proposed method does not need anyextraction step as our preliminary data indicates that the RPA method is compatible with the cell lysis reagent.We plan to achieve our goal by pursuing the following specific aims, namely, (1)(a) Design and optimization ofthe amplification as well as Ztag and reporter incorporation using single-step, one-pot RPA for the detection ofthe pathogenic strains E. coli O157, E. coli O26, and E. coli O121. (b) Evaluation of the binding affinitybetween ZFP and target-incorporated Ztag; (2) Design and development of a flow-strip-based, multiplexDNA/RNA detection platform for the different E. coli strains; (3)(a) Characterization and validation of thecomplete assay using water and food samples. (b) Evaluation of the long-term stability of the flow stripplatform. The proposed research is significant because it will provide a simple and inexpensive multiplexpathogen detection platform that could achieve a global impact due to reducing the economic burden of foodand water borne diseases in the developed world and improving access to detection technologies in low-resource environments.

Daunert, Sylvia; Deo, Sapna K.
University of Miami
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