Nucleic acid-based assays for pathogen detection and identification offer sensitivity, specificity and resolution. Unfortunately, the manipulations for these assays have traditionally relied on nucleic acid isolation and amplification methods poorly suited for use outside of an appropriately equipped laboratory. Nonetheless, a significant need exists for more comprehensive, specific and facile diagnostics for use at the point-of-care. The recent emergence of several isothermal nucleic acid amplification strategies promise to alleviate the need for thermocycling hardware to achieve sensitive nucleic acid detection. Liberated from demanding hardware requirements, nucleic acid amplification becomes suitable for use under circumstances where access to a laboratory infrastructure is limited or unavailable. To enable sensitive, multiplexed detection of isothermally amplified nucleic acids without costly or complex instrumentation, we developed a rapid lateral flow chromatographic approach to DNA microarray fabrication and hybridization. In highly miniaturized embodiments, we refer to as lateral flow microarrays (LFM), the technology enables the detection of 250 amol of nucleic acid analyte in 2 minutes. Coupled with isothermal amplification, the method addresses many of the hurdles to the translation of nucleic acid assays from the laboratory to the field. However, a significant remaining challenge is presented by the need to render a complex biological sample, containing enzyme inhibitors and nucleases, suitable for isothermal amplification. Further, the integration of sample preparation, amplification and detection into a low cost, yet easily used, system is required to realize a facile and robust point-of-care diagnostic tool. The over arching hypothesis of this proposal is that a highly simplified and integrated nucleic acid analysis device can be realized using low cost lateral flow chromatography technologies. Making use of easily fabricated lateral flow devices offers an approach to sample preparation that is facile and familiar to the end user and provides a solid-phase support to capture and concentrate diagnostic targets from confounding sample matrix constituents. Leveraging our prior advances in LFM technology and the availability of sensitive detection schemes compatible with low cost instrumentation, we will develop an integrated nucleic acid analysis system making use of LFM and supporting field deployable sample preparation and amplification methods.
The proposed work will result in the development of a device capable of rapidly detecting nucleic acid sequences in clinical specimens. The suitability of the device for use outside of a laboratory infrastructure will provide a unique ability to conduct informative DNA or RNA-based diagnostic assays at the point-of-care.
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