This Small Business Innovation Research (SBIR) Phase II project proposes the development of novel enzymes (DNA polymerases) and other improvements for rapid detection of food-borne pathogens by DNA detection and amplification (PCR). PCR is a very fast and accurate method of pathogen detection, typically giving results in about a day, instead of several days required to grow and identify pathogens by cultural methods. But some foods, such as chocolate, dairy products, meat, and spices, contain components that inhibit the PCR assay. Current strategies for rapid pathogen testing in these foods include long cultural enrichment steps followed by dilution of inhibitors and/or labor intensive sample preparation (DNA extraction) to remove inhibitors. Inhibition-resistant DNA polymerases and food-specific PCR enhancers represent elegant, high-tech alternatives to dilution or DNA extraction. They could be integrated into existing rapid-detection systems to facilitate rapid accurate testing in inhibitory foods.
The broader impacts of this research are reducing the number and severity of outbreaks of food-borne illnesses in the United States due to early detection of food-borne pathogens. Faster, more accurate detection of pathogens will save time and money for food manufacturers, and reduce the need for costly product recalls. Technology developed here could also extend the disciplines of forensics, where recovery of small amounts of DNA in the presence of a variety of inhibitors is critical, and national defense, where rapid detection of biological agents used as weapons could save lives.
The primary outcome of this research has been products that improve the detection of Salmonella and other dangerous microbes in food. There is an ongoing transformation in the way dangerous microbes are found in foods as we move from growing cells towards using DNA analysis. And DNA analysis is being further improved to make it faster, more accurate, and less expensive. The primary process of DNA analysis is called the polymerase chain reaction (PCR). Yet, PCR can encounter problems if there are substances from the food samples that interfere or inhibit the reaction. Until now, the DNA of the microbe to be analyzed has had to be separated from the food samples prior to PCR. To address this and other problems, our research centered on changing the structure of the enzyme that operates as the catalyst of PCR. We were able to randomly create a variety of enzymes and select the ones that performed well even in the presence of food samples. By reading the amino acid sequence of these modified enzymes, we were able to learn which amino acids are responsible for the improved performance. We filed a patent application that has been made available to the public which details the new technology. The location of the amino acid changes which improve enzyme performance further expands the publicâ€™s understanding of the mechanisms of this important enzyme, which is called Thermus aquaticus, or Taq. The outcome of this research makes possible quicker recalls of infected food products before they become available to consumers.