Multiplexed pathogen detection by on-chip amplification

描述(由调查人员提供)：无论是在临床还是流行病学(公共卫生和环境)环境中，在临床症状出现之前都需要继续检测病原体；特别是对于A-C类药物，这一需求转化为分散的设备和方法，用于检测大量环境样本中的痕量生物。目标病原体的活性、传染性和活/死状态可能是一个重要的指标或要求，要求同时分析同一样本中的DNA和RNA。因此，公共卫生病原体检测的困境对当前的微流控聚合酶链式反应和/或阵列检测设备提出了独特的挑战。然而，溶液相聚合酶链式反应本身在单个样本中可以访问的基因靶标的数量以及与常见的(类似TaqMan的)报告器和分子信标的光学干扰方面受到限制。这项应用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个通用的高通量生物芯片平台，用于同时在芯片上纯化DNA和RNA；芯片上的聚合酶链式反应和逆转录聚合酶链式反应方法，用于超灵敏地检测复杂环境样本中的低丰度核酸；可在中央诊断实验室内外广泛传播和被其他人使用的聚合酶链式反应芯片制造方法；以及针对A-C类病原体的100复合体扩增芯片。我们将通过以下方式实现这些目标：环境样品的自动化亲和分离方面的长期工作；Argonne独特的三维凝胶垫微阵列，以在溶液相空间有序阵列中固定亲和探针；支持凝胶内热循环和核酸放大的新型(专有)凝胶组合物；以及正在进行的用于内场生物芯片成像和分析的国防部仪器开发活动。我们将在纯培养和修改后的气溶胶、地表水(河流、沼泽、池塘)和土壤样本中验证BSL-2细菌病原体DNA和RNA靶标的技术。在修改后的环境样本中成功展示高度复合的RT-PCR芯片，将为开发分布式诊断系统以快速检测和表征自然环境中的病原体，以及在生理/临床样本中进一步验证原型系统奠定基础。

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.