Development of an In-situ Capable Method for Detecting Pathogenic Bacteria in the Alabama

F. PROJECT OVERVIEW/SUMMARY: Proper stewardship of Alabama's tremendous water resources relies on the amount of data and tools available to formulate and execute management strategies. In other words, the availability of a miniaturized in-situ pathogen detection system will have enormous impact on the way we manage the contamination of our water resources. It will open up numerous possibilities in terms of monitoring, tracing and rectifying the contamination source. However, the development of such an in-situ pathogen detection system is contingent on the availability of a rapid, accurate, and economic detection technology. For this reason, we developed a rapid, accurate, in-situ capable technique for the detection of pathogens (E. coli O157:H7) in water at levels as low as 100 organisms per mL. The development of the in-situ capable pathogen system consists of three phases as shown in Figure 1 below. Figure 1. The overall flow scheme of our research objectives in each task for three phases. Phase 1 and 2 have been completed in FY 2010-2011 and Phase 3 is currently being implemented in FY 2012. Phase 1 (FY2010) was a proof-of-concept study in a laboratory setup to develop a novel, in-situ capable technique for rapid, accurate, and sensitive pathogen detection in water environments. The PHASE 1 (Completed FY2010) Method development and proof of concept PHASE 2 (Completed FY2011) Intermediate development for field-worthiness of components Task 1. Assay development targeting synthetic DNA Task 2. Detection of pathogenic bacteria and method optimization Task 3. Assay validation by a conventional method Task 1. Inhibition characterization of environmental samples Task 2. Field compatibility of operation parameters such as incubation, mixing, stability, and DNA pre-treatment PHASE 3 (In progress) Development of system components and integration Task 3. Preliminary experiments on DNA hybridization in a microfluidic environment Task 1. Development of inline on-chip mixing and hybridization Task 2. Development of inline magnetic trap Task 3. System integration on briefcase platform 3 methodology is based on the specific DNA hybridization using our custom configured multi-functional nanoparticle labels. Our unique hybridization method was investigated for its ability to quantitatively detect pathogens in the forms of synthetic linear DNA (Task 1) and genomic DNA from bacterial culture (Task 2). Both tasks determined the quantitative parameters for pathogen DNA detection such as linearity (correlation coefficient and range of quantification), assay sensitivity (detection limits), specificity (mismatches), and rapidity of assay (reaction kinetics) in the laboratory. In Task 3, conventional plate counting …