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The goal of this proposal is to develop a portable diagnostic kit that will allow rapid (~2 hours), sensitive (100 CFU per ml) and selective detection of ribosomal RNA of Brucella in dairy products. <P>Specific objectives are : 1. Develop sample preparation units enabling isolation of bacteria from raw milk and the isolation of RNA from cells. 2. Refine Notre Dame's nanomembrane RNA sensors and optimize its ability to selectively detect Brucella neotomae RNA---a surrogate for pathogen Brucella within a heterogeneous RNA mixture extracted from bacteria cells 3. Demonstrate proof-of-concept in a microfluidic platform to detect ribosomal Brucella against real food samples. <P>The milestones are Year 1: Demonstrate efficient RNA extraction from bacterial cells from raw milk using sample preparation system Year 2: Develop nanomembrane sensor based on UV-patterned polymer and immobilize RNA probes with repeatable and optimal density Year 3: Demonstrate a proof-of-concept membrane sensor platform to detect against real food samples.
Non-Technical Summary:<br/>
Foodborne diseases are one of the most common causes of morbidity and mortality worldwide, and have significant economic consequences with financial losses accounting to billions of dollars to food industry. Over the past decade, the noticeable increase in foodborne illnesses due to the bacterial contamination of foods have been reported with increasing illnesses resulting from consumption of fresh fruits and vegetables and furthermore, there is continued threat of transmission of emerging pathogens such as Brucella sp., Mycobacterium paratuberculaosis, Clostridium difficile and many others through food . Recent CDC data indicate that there are about 48 million illnesses, 128,000 hospitalizations and 3,000 deaths occur annually in the US due to foodborne infections. Existing methods for detecting Brucella: Although the "gold standard" for the diagnosis of brucellosis is isolation of Brucella bacteria, but its isolation is time- and resource-intensive; it requires level 3 biocontainment facilities and highly skilled technical personnel to handle samples and live bacteria for eventual identification and biotyping. Further, isolation of Brucella requires prolonged incubation time (can be up to 2 weeks) and is not always successful, PCR-based detection (especially real-time PCR assays) is becoming more accepted. To date, at least 400 reports have been published dealing with various PCR-based methods for Brucellosis detection. But the current PCR-based Brucella detection are laborious, expensive and require specialized laboratory facilities and technician and hence not suitable for on-field application. Hence, development of a smart point-of-care rapid assay system is necessary to control the spreading of this bacterium. Thus, there is a demand to develop a rapid, portable, selective, and PCR free RNA sensing platform for Brucella that can be easily employed for on-field application.
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Approach:<br/>
The portable diagnostic kit is superior to current bacteria detection method in that it is rapid (~2 hours) and does not require fluorescent labeling or PCR amplification. The rapidity is due to concentration of analyte molecules from a flowing solution by dielectrophoretic and depletion action provided by an electric field through a nanoporous membrane. Selectivity is afforded by the complementary probes functionalized on the surface of the membrane and robust detection without spurious electrochemical reaction is achieved by monitoring the change in ion current due to the presence of the hybridized RNA. The membrane biochip is very economical to produce and the microfluidic instrumentation is portable.