SBIR-STTR Award

Analysis of nucleic acids represents a well established molecular diagnostic test offering a highly specific detection of pathogens causing infectious diseases. Most clinical methods are based on the process of amplifying DNA and/or RNA molecules using polymerase chain reaction (PCR). The proposing team has recently developed another scheme for efficient isothermal amplification of DNA; the ramification amplification (RAM). This isothermal amplification is derived from primer extension, strand displacement, and the propagation of multiple ramification (branching) points. This technology utilizes a capture probe for target DNA or RNA isolation and a circularized RAM probe for subsequent amplification. Isothermal methods of DNA amplification have inherent advantages compared to the PCR method because there is no need to perform thermal cycling and can be easily packaged into a portable detection system. The proposed project combines two technologies, RAM amplification and electronic microarray technology where an electric field is used to assist and accelerate the DNA amplification process. The project is a joint effort in combining two compatible technologies and will leverage earlier efforts in RAM amplification and development of a portable DNA microarray detection system. The main advantage of the proposed approach is that a highly selective and sensitive RAM amplification will be integrated on the electronic microarray which will assure high level of multiplexing and rapid detection. Potential applications are envisioned in portable and/or point of care systems for detection of pathogens, infectious diseases and/or biological agents and emerging pathogens

Recent events such as the spread of swine flu and potential future detrimental mutations of influenza viruses point to an urgent need for a point-of-care influenza typing system; particularly because over 90% of hospitals and smaller clinical labs in the U.S. cannot perform DNA-based diagnostic analysis. To enable a quick response to a potential influenza outbreak, it is desirable to have a fast, accurate diagnostic method capable of simultaneously typing and subtyping influenza viruses. Innovative solutions are needed in sample preparation, DNA/RNA extraction, microfluidics, DNA amplification methods as well as instrument miniaturization to make bacterial and/or viral point-of-care molecular diagnostics a reality. This project leverages a feasibility of on-chip, real time rolling circle amplification of DNA on an electronic microarray platform demonstrated in Phase I with our efforts in miniaturization of DNA detection and development of platforms for integration with the sample preparation. The following innovative solutions form the basis of the proposed technology:(i) novel real-time fluorescence detection of (branched) rolling circle amplification of DNA on the electronic microarray platform; (ii) a disposable cartridge that incorporates a unique fluidics enabling magnetic bead DNA/RNA extraction, reverse transcription, DNA amplification and microarray detection; (iii) low cost, screen printed carbon electrodes-based microarray for electric field-assisted acceleration of DNA hybridization. The Phase II project specific aims will demonstrate the feasibility of the technology that will consist of: (1) Optimization of real-time rolling circle amplification (RCA) conditions on the carbon-based electronic microarray; (2) Development and optimization of influenza virus genotyping assay based on hemagglutinin (HA) and neuraminidase (N) typing; (3) Development of sample preparation chemistry for influenza typing assay and incorporation onto a disposable cartridge using magnetic bead based separation of RNA/DNA, reverse transcription and RCA amplification; (4) MDx system development and adaptation to influenza assay; and, (5) Validation of the influenza assay using spiked and clinical (archived) samples.

Public Health Relevance:
The recent spread of the H1N1 swine-origin influenza virus and potential detrimental mutations of influenza viruses point to an urgent need to develop point-of-care systems that will be capable of typing influenza viruses. Molecular diagnostics methods providing accurate typing of a virus will ensure a quick and appropriate response and enable improved management of patients and limit transmission of viruses. Standard techniques for viral typing are mostly based on PCR or real-time RT-PCR methods that are complex and require several hours to perform the analysis. This project brings a new real-time method for detection of DNA based on an extremely rapid rolling circle amplification of DNA/RNA targets on the electronic microarray platform. In the proposed project, the instrument and the assay will be fully developed and provide a low-cost, portable solution to diagnose influenza and/or other infectious diseases.

Thesaurus Terms:
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