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，都持续需要在临床症状出现之前检测病原体;特别是对于A-C类物剂，这种需要转化为用于检测大体积环境样本中痕量有机体的分散式设备和方法。目标病原体的活力、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的困境对当前的微流体PCR和/或阵列检测装置提出了独特的挑战。然而，溶液相PCR本身在单个样品内可以访问的基因靶标的数量以及与常见（TaqMan样）报告基因和分子信标的光学干扰方面是有限的。本申请的目的是克服这些缺陷，并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：开发一个通用的高通量生物芯片平台，用于同时进行芯片上DNA和RNA纯化;芯片上PCR和RT-PCR方法，用于对复杂环境样本中的低丰度核酸进行超灵敏检测; PCR芯片制作方法，可在中央诊断实验室内外广泛传播和供他人使用;和针对A-C类病原体的100重扩增芯片。我们将满足这些目标，利用长期的工作，在自动化亲和分离环境样品;阿贡的独特的三维凝胶垫微阵列，在一个溶液相，空间有序的阵列，新的（专有）凝胶组合物，支持凝胶内热循环和核酸扩增;和正在进行的国防部仪器开发活动的内场生物芯片成像和分析。我们将在纯培养物和改良气溶胶、地表水（河流、沼泽、池塘）和土壤样品中验证BSL-2细菌病原体DNA和RNA靶标的技术。一个高度多重RT-PCR芯片在一个修正的环境样品中的成功演示将奠定基础，为分布式诊断系统的发展，在自然环境中的病原体的快速检测和表征，并在生理/临床样品的原型系统的进一步验证测试。

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.