Multiplexed pathogen detection by on-chip amplification

描述（由研究者提供）：无论是在临床还是流行病学（公共卫生和环境）环境中，都需要在出现临床症状之前检测病原体；特别是对于A-C类药剂，这种需要转化为用于检测大量环境样品中微量生物的分散装置和方法。目标病原体的生存力、感染性和活/死状态可能是一个重要的指标或要求，需要同时分析同一样品中的DNA和RNA。因此，公共卫生病原体检测困境对当前的微流控PCR和/或阵列检测设备提出了独特的挑战。然而，溶液相PCR本身在单个样品中可以访问的基因靶标数量和普通（taqman样）报告器和分子信标的光学干扰方面受到限制。本申请的目的是克服这些不足，开发一种集成的、三维凝胶垫样品纯化和扩增/检测芯片，以检测环境中的A-C类病原体。具体目标包括开发一种通用的高通量生物芯片平台，用于同时进行芯片上DNA和RNA的纯化；芯片上PCR和RT-PCR超灵敏检测复杂环境样品中低丰度核酸的方法；可在集中诊断实验室内外广泛传播和供他人使用的PCR芯片制造方法；以及针对a - c类病原体的100倍扩增芯片。我们将通过利用长期以来在环境样品的自动亲和分离方面的工作来实现这些目标；Argonne独特的三维凝胶垫微阵列将亲和探针固定在溶液相，空间有序阵列中；支持凝胶内热循环和核酸扩增的新型（专有）凝胶组合物；以及正在进行的国防部仪器开发活动，用于内场生物芯片成像和分析。我们将在纯培养物和改性气溶胶、地表水（河流、沼泽、池塘）和土壤样品中对BSL-2细菌病原体DNA和RNA靶点进行验证。高复用RT-PCR芯片在改良的环境样品中的成功演示，将为分布式诊断系统的发展奠定基础，用于快速检测和表征自然环境中的病原体，并进一步验证生理/临床样品中的原型系统。

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.