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Saint Louis, MO, United States

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 105.91K | Year: 2010

DESCRIPTION (provided by applicant): We propose to develop a highly simplified and improved method of detecting RNA for use in clinical tests and for scientific research by enabling the RT-PCR amplification of nucleic acids directly in whole blood, serum, plasma, and cell lysates. We propose a dual approach. We will work with two of our blood inhibition Taq mutants combined with a viral reverse transcriptase enzyme in the presence of a specially developed enhancer. In addition, we will make amino acid substitutions to our blood inhibition mutants to render them competent in reverse transcription. The sensitivity of the new technology will drive the evaluations. The method also includes development and optimization of buffers that are compatible with both the RT and DNA polymerase activities, as well as reaction additives that relieve the inhibition and enhance the RT-PCR performance in crude specimens. The tests will begin with mimic samples composed of RNA mixed with blood, serum, and plasma. Once optimized, the method will be applied to clinical RNA pathogen detection and mRNA expression assays in crude samples. The clinical RNA virus pathogens will include HCV and GBV. Comparisons will be made to the standard RNA detection protocol, which requires the RNA to be purified from the sample prior to detection. The focus of the novel method will be to increase the sensitivity of detection to match or exceed the sensitivity of the standard method. Until now, the diagnosis of infectious diseases and genetic disorders has required costly and time-consuming procedures. The standard protocol requires RNA purification prior to RT-PCR which may reduce the quantity of RNA before cDNA is produced. The proposed novel technology not only introduces a significant reduction in cost, but also solves technical problems irrespective of cost. The proposed method would provide improved accuracy, efficiency, and lower cost of RNA detection directly in whole blood or blood fractions samples and cell and tissue lysates. The benefit to the public is by improved and more reliable detection of RNA pathogens in clinical tests and advanced means of measuring mRNA expression in crude samples at a reduced cost. PUBLIC HEALTH RELEVANCE: Many viruses that are harmful to humans, such as hepatitis, HIV, and influenza, are based in RNA, not DNA. To determine if a patient has a harmful RNA virus, a blood sample is often drawn then sent to a lab to use a process called RT-PCR to find the virus. Until now, for RT- PCR to work, the RNA must first be extracted from the blood which can present problems and is expensive, but our proposed method can find RNA directly in blood.

Agency: NSF | Branch: Standard Grant | Program: | Phase: | Award Amount: 500.00K | Year: 2011

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.

Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 142.50K | Year: 2009

This Small Business Innovation Research (SBIR) Phase I project proposes the improvement of real-time PCR, a DNA-based rapid-detection method, for detection of food-borne pathogens such as Salmonella. Current methods are inadequate to accurately detect pathogens in foods such as chocolate, soft-cheese, and milk, which are inhibitory to PCR. These foods present a challenge due to their interference with the sensitivity of the assay. Longer, more labor-intensive tests are required for these foods, as inaccurate detection can lead to potentially deadly false negatives. The proposed improvements will shorten and simplify pathogen detection for these foods. The novel inhibition-resistant mutant enzymes and enhancers can to applied to PCR, and simplify other stages of current protocols such as cultural enrichment (an incubation period usually required for pathogen detection) and sample preparation. These improvements may also allow for more rapid, accurate testing for a broad range of foods, not only those foods known to be inhibitory to PCR. The broader impacts of this research include a safer food supply, due to the faster, more accurate detection of food-borne pathogens. This will allow food producers and manufacturers to more quickly respond to any discovered contamination, and facilitate immediate recalls of infected food. For routine tests, these innovations will give testing labs accurate and reliable results, while saving them time, labor, and money.

DNA Polymerase Technology, Inc. | Date: 2013-10-16

Provided herein are mutant polymerase enzymes resistant to inhibitors encountered in Polymerase Chain Reactions (PCR). Also provided are nucleic acids or constructs encoding isolated polypeptides having polymerase activity. Also provided are kits useful for PCR containing isolated polypeptides having polymerase activity or isolated nucleic acids encoding such.

The present invention generally relates to detection of a target nucleic acid in standard PCR, real-time PCR, RT PCR, and real-time RT PCR. One aspect of the invention provides mutant DNA polymerase enzymes that are resistant to PCR inhibitors, such as dye, blood, and soil. Another aspect of the invention provides for methods of real-time PCR assays using mutant DNA polymerase enzymes resistant to PCR inhibitors with samples containing dye, blood, and/or soil. Another aspect of the invention provides for methods of standard PCR assays using mutant DNA polymerase enzymes resistant to PCR inhibitors with samples containing blood and/or soil.

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