1a. Objectives (from AD-416):
1: Develop and integrate operational technologies to rapidly and effectively concentrate viable target cells from food matrices in a self-validating system into an automated instrument. 1A: Integrate technology platforms that we have developed and individually tested into a usable technology for detecting L. monocytogenes in less than 8 h (time-to-result). 1B: Integrate technology platforms, currently being developed in our laboratory, into a usable technology for detecting Salmonella . 1C: Integrate technology platforms into a usable technology for detecting STEC. 2: Develop, evaluate, and adopt novel technologies for rapid detection, identification, and quantification of viable and non-viable target microorganisms. Research areas to be addressed include microfluidic biochips, optical light scattering technology, bacteriophage sensors, and Raman spectroscopy. 2A: Microfabricate and characterize microfluidic biochips that will direct, concentrate, and quantify living microorganisms using micro- and nano-scale electrical, mechanical, and optical methodologies. 2B: Develop light scattering technologies for rapid and high throughput detection and identification of pathogenic bacteria based on unique scattering signatures generated by concentrated colonies. 2C: Develop bacteriophages carrying reporter genes for the detection of E. coli O157:H7 and other foodborne pathogenic bacteria. 2D: Develop a highly sensitive enhanced Raman spectrosensor for field-deployable and routine benchtop in-lab identification of foodborne pathogens.
1b. Approach (from AD-416):
The approach focuses on four main components including separation, detection, identification, and quantification of target microorganisms from food matrices.
3. Progress Report:
To prevent outbreaks of foodborne illness, food regulatory agencies and the food industry need rapid, sensitive, and specific methods to check for the presence of harmful bacteria (pathogens) in food. Our approach to development such methods involves novel bioseparation technology to separate and concentrate pathogens from foods via sequential filtration (a 1000-fold concentration of cells in less than 30 minute) and various methods to detect and quantify foodborne pathogens. Two prototype filtration instruments were built this year. The filtration device was used to separate and concentrate natural flora and pathogens from water and foods (chicken, milk, cookie dough, and vegetable wash water) and, when coupled to polymerase chain reaction (PCR) analysis, pathogen detection was completed within 6 hours. A variety of platforms appropriate for detection of the concentrated pathogens were studied. One method uses a microfluidic biochip that can further concentrate bacteria, supports bacterial growth, and breaks the bacteria open to release their DNA to detect pathogen-specific genes. In addition, a novel method was developed to rapidly heat sub-nanoliter droplets on the biochips, to facilitate ultra-rapid (< 10 min) PCR assays. This year developments in loop mediated isothermal amplification (LAMP) enabled multiplexed screening of virulence genes of L. monocytogenes, E. coli, and Salmonella on the chip. A second system called BARDOT (Bacterial Rapid Detection using Optical Scattering Technology) uses light scattering techniques to differentiate and classify bacterial colonies grown on Petri-dishes. Major hardware and software updates supported the development of a portable instrument, and a prototype was delivered to USDA for further testing and application development. The BARDOT technology was augmented with different optical methods (Raman spectroscopy and a multispectral interrogation) and preliminary analyses of E.coli and Listeria are underway. Efforts are ongoing to validate pathogen identification by BARDOT in real world produce samples through DNA sequence verification. A third system employs an E. coli O157:H7-specific phage that was genetically engineered to cause the target bacteria to produce a bright yellow color if infected. This year methods were developed to encapsulate the phage into a pill format using commercially available polymers to enhance shelf-life and facilitate their addition to selective enrichments. Efforts are ongoing to expand the reporter phage specificity to other Shiga toxin-producing E. coli. Also, methods employing fluorescent immunoassays in combination with magnetic concentration enabled rapid (2 hours) and sensitive (5 CFU/mL) detection of E.coli O157:H7, Salmonella, and L. monocytogenes. Finally, as part of larger study, DNA fingerprinting of over 800 L. monocytogenes isolates from retail delis representing diverse geographic regions was completed.