A Universal Nucleic Acid Recognition Platform for Detection of Pathogenic Bacteria

The goal of this research is to develop a low-cost and highly sensitive and selective point-of-care (POC) device that will detect disease-causing bacteria at room temperature. The success of the project requires differentiation between harmless and disease-causing bacteria, and is possible by detection of tiny differences in bacterial RNA (genetic material). The proposed research presents a sensor device with a simple design that consists of a universal nucleic acid probe and two intermediary strands of nucleic acids organized in such a way that changes in their natural association can be used to measure the presence the disease-causing bacteria. Broader impacts for education and the community include the development of the outreach program called GRoW uP in the Central Florida area.<br/><br/>Current approaches for detecting pathogens, such as nucleic acid amplification tests (NAATs), offer high sensitivity, but often produce false-positive results, and require expensive instruments and trained personnel. Furthermore, these approaches cannot differentiate between the point mutations that distinguish the RNA of the pathogenic from the non-pathogenic bacteria. This is especially difficult at ambient temperatures and for RNA that forms secondary structure. The goal of this proposal is to develop a specific and sensitive universal point-of-care (POC) platform for the rapid and accurate detection of pathogenic bacteria. The approach uses a binary probe with high sequence recognition specificity that is necessary to differentiate point mutations and is amenable to analysis of folded RNA. The new methodology will exceed the performance of current state-of-the-art approaches for RNA sensing in the following aspects: (i) it will exhibit zero false-negative responses, thus improving sensitivity and reducing limits of detection (LOD); (ii) it will enable accurate recognition of point mutations at ambient temperatures even in structured (folded) RNA and DNA; (iii) it will enable detection of multiple analytes using a single universal probe in a re-usable format and thus reduce the assay cost. The proposed work builds upon the PI?s recent improvements on the self-assembling DNA 4J fluorescent sensor by converting the system to an electrochemical biosensor (4J E-biosensor). Advantages of the 4J E-biosensor include the capability of performing multiple tests with zero signal background and the potential for on-site testing while offering fast, simple, low-power, portable, and inexpensive detection. The novel sensor platform will advance the ability to detect ribosomal RNA and thus, have unprecedented impact in environmental, health diagnosis, national defense and food safety applications. The interdisciplinary nature of the proposed work will expose students to different areas of chemistry, including electrochemistry, biochemistry, computational simulations, sensing applications, and nanomaterial sciences. The PI proposes to use this research as a motivation to continue and expand her self-developed GRoW uP program. The program will provide research opportunities to high school students and teachers through partnerships established with Orange Public School District. The PI will also implement an outreach program in conjunction with the Orlando Science Center and the Seminole County Library to impact hundreds of young people across multi-ethnic Orlando and Central Florida. Dissemination of the results from this investigation will be through publications in high-impact peer-reviewed journals and national and international conferences.