A Multiplexed Assay for Identifying Foodborne Bacteria

Our goalsare to develop a low-cost field-deployable instrument for identifying foodborne bacteria. The research will empower state-federal food and agriculture researchers for early surveillance of bacteria contaminants in the field. Our plan is to identify foodborne bacteria by (i) synthesizing Buckyballs with specific nucleic acid sequences that are specific to a unique region of the 16S ribosomal RNA (rRNA) region for each bacterium, and (ii) optimizing the design of multiplexed assays using a microfluidic system for field-based deployment. The technology also allows identification of live bacteria. Aim 1: To develop, optimize, and validate probes for each foodborne bacterium: Probe complexes will be synthesized for 11 foodborne bacteria that have been selected from the CDC study. Synthesized probes will be profiled and validated for hybridization, crosstalk, and limits of detection. These probe complexes are of the order of 20-30mers. Every attempt will be made to optimize for the limit of detection (LOD) of a bacterium with a single fluorophore. Synthesis of probe complexes will be complemented with the necessary controls to determine sensitivity, specificity, and validation.Aim 2: To evaluate alternative designs of a microfluidic system for multiplexing: Several design concepts will be fabricated, tested, and validated for reliability and sensitivity. The basic design consists of a disposable low-cost microfluidic system, which includes a single source and radially distributed sinks connected with channels to the source. A unique DNA-RNA complex with the fluorescent reporter (per Aim 1), corresponding to the 16S rRNA of each bacterium, will be printed at each sink. Some of the sinks will strictly be for positive and negative controls. The microfluidic design is flexible to allow increased concentration of the bacteria (as an alternative plan); thus, increasing the signal to noise ratio for the readout. A number of design configurations will be to quantify sensitivity and limit of detection (LOD) by an external reader.The deliverables will be (i) at least one major publications, (ii) probes for identifying a subset of foodborne bacteria, and (iii) a preliminary design of amicrofluidic system to support imaging of foodborne bacteria.