# Portable, Rapid, Multiplexed Flow strip Test for Bacteria

> **NIH NIH R01** · UNIVERSITY OF MIAMI SCHOOL OF MEDICINE · 2020 · $303,163

## Abstract

PROJECT SUMMARY
Cost-effective on-site assay technologies for pathogen detection are sorely lacking, even though they are
essential 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 billion
in the US. One of the most common strategies for foodborne pathogen detection is laboratory-based
polymerase chain reaction (PCR) of a single pathogen type that takes about 24 h followed by confirmation
using culture-based methods that take a minimum of 1 week. Additionally, a majority of techniques reli only on
DNA whereas RNA is a better indicator of viable bacteria. Ideal technology should target detection of multiple
pathogens through the detection of specific nucleic acid in a portable on-site detection platform. However, the
detection 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 significant
challenge. To that end, our overall goal is to develop a flow-strip multiplex pathogen detection platform that will
fulfill the following criteria: simple sample preparation, rapid multiplexed DNA/RNA target amplification and
incorporation of reporter in a single pot, portable platform with colorimetric detection for on-site and
inexpensive monitoring. Here, we propose to use recombinase polymerase amplification (RPA) and reverse
transcriptase-RPA to incorporate unique zinc-finger protein (ZFP) binding motifs (referred to as “Ztags”) and
reporter moieties into the amplification product from nucleic acid targets of interest in order to detect the
presence 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 data
demonstrated that we can incorporate both Ztag and reporter into the target nucleic acid using the RPA
method and detect as low as 10 copies of target. Additionally, the proposed method does not need any
extraction 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 of
the amplification as well as Ztag and reporter incorporation using single-step, one-pot RPA for the detection of
the pathogenic strains E. coli O157, E. coli O26, and E. coli O121. (b) Evaluation of the binding affinity
between ZFP and target-incorporated Ztag; (2) Design and development of a flow-strip-based, multiplex
DNA/RNA detection platform for the different E. coli strains; (3)(a) Characterization and validation of the
complete assay using water and food samples. (b) Evaluation of the long-term stability of the flow strip
platform. The proposed research is significant because it will provide a simple...

## Key facts

- **NIH application ID:** 9912174
- **Project number:** 5R01GM127706-03
- **Recipient organization:** UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
- **Principal Investigator:** Sylvia Daunert
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $303,163
- **Award type:** 5
- **Project period:** 2018-08-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9912174

## Citation

> US National Institutes of Health, RePORTER application 9912174, Portable, Rapid, Multiplexed Flow strip Test for Bacteria (5R01GM127706-03). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/9912174. Licensed CC0.

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