Staphylococcus aureus is a highly successful bacterium that causes a wide range of local and systemic diseases. In the face of selective pressure, it evolves efficiently to develop drug-resistant strains and strains with a variety of virulence factors, both in hospital and in community settings. The long-term goal of the proposed work is to develop and commercialize a compact real-time PCR chip to detect and genetically profile S. aureus from clinical samples. The prototype chip will be the size of a cover-slip, and will be derivatized with an array of self-reporting probes. Because this array will utilize multiple probes for multiple target regions on each amplicon, an exceptionally high level of accuracy will be achieved, along with the ability to localize mutations and deletions within each amplicon. Future versions of the chip will have multiple wells, each coated with arrays of self-reporting probes. In a variety of potential manifestations, the chip will provide a versatile component for integration into a wide range of diagnostic platforms.
The specific aims of this Phase I application are: 1) To optimize surfaces for the functional attachment of bimolecular beacons (BiMBs) for variable temperature applications. We now routinely obtain 10-100 fold fluorescence enhancements of surface-bound BiMBs upon interaction with target. We will optimize our surface chemistries to facilitate monitoring of BiMBs over the full range of temperatures necessary for on-chip RT-PCR. 2) To develop a thermally controlled chip that uses self-reporting YES/NO switches in order to monitor target antibiotic resistance and toxin genes. The chip will comprise a surface-phase self-reporting array in contact with a liquid phase containing target sequences, confined within a thermally controlled chamber that will be imaged in real-time by fluorescence microscopy. 3) To develop a prototype on-chip PCR platform that monitors target sequence amplification during the course of RT-PCR, and thereby functions to identify S. aureus, and to determine if it is methicillin resistant, and whether it harbors the TSST-1 and PVL genes. The prototype RT- PCR chip will be designed with multiple fixed BiMB switches directed against different regions for each target gene. The chip results will be evaluated against phenotypic and solution-based PCR tests that have been well- validated in clinical settings. In phase II of this project, the focus will be on pre-commercial development of rapid, sensitive, specific and reliable S. aureus diagnostic chips that use microliter volumes of a variety of clinically relevant sample types.
The public health relevance of this work is that the proposed diagnostic device will provide an inexpensive, robust, and precise means of rapidly diagnosing and profiling S. aureus infections with respect to antibiotic resistance and to a variety of virulence factors. Whereas current S. aureus diagnostic methods generally take from 24 to 48 hours for a preliminary characterization, the proposed method will take no more that two hours, and will provide a comprehensive characterization of unparalleled diagnostic accuracy. ? ? ?
| Braunlin, William; Völker, Jens; Plum, G Eric et al. (2013) DNA meter: Energy tunable, quantitative hybridization assay. Biopolymers 99:408-17 |
