Escherichia coli O157:H7 is an important cause of foodborne diseases with symptoms of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. Salmonella spp. are widely distributed in nature and are a major cause of foodborne illness with symptoms of nausea, vomiting, and diarrhea in the United States. Accurate and fast detection method for foodborne pathogens from various food samples has always been a desirable and important goal that has attracted scientists from many research areas. Quantum dots (QDs) are a family of nanosized particles comprised of a few thousands atoms, with typical size 1 to 10 nm in radius. QDs have long-term stable photostability, high quantum yield, broad absorption spectra and narrow, symmetric emission. QDs demonstrate high signal-to-noise ratio, allowing for much improved sensitivity of QD-based detection methods. In this study, a combination of 1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride (EDC) and protein A was used to form a crosslinker between antibodies and QDs. In addition, bead-free immunomagnetic separation (IMS) was tested to isolate target E. coli O157:H7 and Salmonella from ground beef products.
The objective of this study was to use a bead-free IMS method to isolate E. coli O157:H7 and Salmonella from ground beef and QD-labeled antibodies as a reporter to detect the presence of E. coli O157:H7 or Salmonella.
Green QDs and orange QDs were synthesized. The chemical and physical properties and the concentration of the QDs were tested and optimized. Twenty-five microliters (for green QDs) or 28 μl (for orange QDs) of EDC and 1.5 mg protein A were added to each 100 μl green or orange QDs, respectively. After a 2-h incubation, 100 μg of E. coli O157:H7 antibodies and 100 μg of Salmonella antibodies were added to the QD-EDC-protein A complex, followed by an additional 2-h incubation at 4oC. Antibody-QD conjugates were made and stored at 4oC for usage. The entire bead-free QD-facilitated pathogen detection method contained three steps: target cell isolation, target cell labeling, and target cell detection.
Additional step:
If the concentration of target cells in ground beef was lower than 106 CFU/g, one additional step consisting of a 24-h enrichment in tryptic soy broth supplemented with 0.5% yeast extract (TSBY) was added before step 1.
This novel bead-free QD-facilitated detection method demonstrated a high fluorescence strength compared to traditional IMS methods when the target cell concentration were equal. This indicated that the bead-free method have high sensitivity for cell detection. This detection method was applied to pure cultures, it detected as low as 10 CFU/ml target pure culture and 106 CFU/g E. coli O157:H7 and Salmonella from ground beef simultaneously. With the incorporation of a 24-h enrichment step, this method can detect as low as 10 CFU/g Salmonella cells from artificially contaminated beef.
Current methods of detecting and differentiating among important pathogens in beef products are limited in their speed and sensitivity. Most sensitive detection methods, such as PCR, are lab-based, which means the samples collected from the beef processing plants have to be transferred to a lab before any analysis can be done. By using QDs coupled with bead-free IMS isolation, we believe that this is the first step for future development of an on-site detection device. QDs are very stable and can be stored in room temperature for up to a year without losing their physical and chemical properties. This makes the on-site detection possible, because their flexibility and stability in any environment allows inspectors to carry them around. Successful realization of this study and the potential continuation of this study will provide a new sensitive and rapid tool to ensure the safety of beef, which in turn, will increase consumer confidence and the market potential of beef products.