Multiplexed pathogen detection by on-chip amplification

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DESCRIPTION (provided by investigator): Whether in clinical or epidemiological (public health and environmental) settings, there is a continued need to detect pathogens before the onset of clinical symptoms; for Category A-C agents in particular, this need translates into de-centralized devices and methods for detecting trace organisms in large-volume environmental samples. Viability, infectivity and live/dead status of the target pathogen may be an important indicator or requirement, requiring the simultaneous analysis of DNA and RNA in the same sample. Thus, the public health pathogen detection predicament presents unique challenges to current microfluidic PCR and/or array detection devices. Solution-phase PCR by itself, however, is limited in the number of gene targets that can be accessed within a single sample and optical interference with common (TaqMan-like) reporters and molecular beacons. The objective of this application is to overcome these deficiencies and develop an integrated, 3-dimensional gel pad sample purification and amplification/detection chip to detect Category A-C pathogens in the environment. Specific aims include developing a common, high-throughput biochip platform for simultaneous, on-chip DNA and RNA purification; on-chip PCR and RT-PCR methods for ultra-sensitive detection of low-abundance nucleic acids within complex environmental samples; methods for PCR chip fabrication that can be widely disseminated and used by others, within and beyond centralized diagnostic laboratories; and a l00-plex amplification chip targeting Category A-C pathogens. We will meet these objectives by taking advantage of long-standing work in automated affinity separations for environmental samples; Argonne's unique 3-dimensional gel-pad microarrays to immobilize affinity probes in a solution-phase, spatially ordered array; new (proprietary) gel compositions that support within-gel thermal cycling and nucleic acid amplification; and on-going DoD instrument development activities for infield biochip imaging and analysis. We will validate the technology on BSL-2 bacterial pathogen DNA and RNA targets in pure culture and amended aerosol, surface water (river, marsh, pond) and soil samples. Successful demonstration of a highly multiplexed RT-PCR chip in an amended environmental sample will lay the foundation for the development of distributed diagnostic systems for the rapid detection and characterization of pathogens in the natural environment, and further validation testing of the prototype systems in physiological/clinical samples.