SBIR Phase I: Low-cost Detection and Enrichment of Nucleic Acids by Interfacing with Commercially Available Cell Sorters

SBIR 第一阶段：通过与市售细胞分选仪连接来低成本检测和富集核酸

• 批准号: 1447889
• 负责人: Adam Sciambi
• 金额: $ 15万
• 依托单位: Mission Bio, Inc.
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1447889&HistoricalAwards=false
• 关键词: SBIR; Phase; Low; cost; Detection

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) project will be to enable affordable detection and enrichment of rare DNA or RNA molecules from a large background population. Currently, such detection is performed on expensive, dedicated instruments that have low sensitivity and are not able to enrich. The proposed strategy is to encapsulate the fluorescently-identified target molecules in thin oil shells so that they mimic cells, and then process them on sensitive and high-throughput cell-sorting instruments that are readily available. By offloading the detection and enrichment to such machines, all that is required by researchers is a simple tool that encapsulates the molecules in thin oil shells. Applications afforded by high sensitivity and enrichment are numerous, from rare pathogen or cell detection to target capture of uncommon mutant sequences with the goal of downstream sequencing. Between the reduced cost and expansion of capabilities, this approach has the potential to become a widely adopted method.This SBIR Phase I project proposes to enable high-throughput digital PCR (dPCR) at a dramatic cost reduction by interfacing with existing flow cytometers (FC). The main research objectives center on characterizing and optimizing the dPCR droplets for use on a wide variety of FC instruments originally intended for cell sorting. First, the biological, chemical, mechanical, and optical properties of the dPCR droplets will be optimized to guarantee compatibility with different FC systems while maximizing earlier PCR efficiency and specificity. Next, serial dilutions of target sequences will be run to test and improve detection thresholds. Those same samples also will be benchmarked against currently commercially available dPCR systems. In tandem, the droplet-on-FC system will be used in a research collaboration to quantitate and characterize HIV latency in an infected cell population. Initial results with FC-processed droplets are promising, as FC systems are already remarkably flexible due to the range of cell types they must be capable of accepting.

这项小型企业创新研究（SBIR）项目的更广泛的影响/商业潜力将是实现来自大型背景人群的稀有DNA或RNA分子的负担得起的检测和富集。当前，此类检测是对昂贵的专用仪器进行的，这些仪器的灵敏度低并且无法丰富。提出的策略是将荧光识别的靶标分子封装在薄油壳中，以使它们模仿细胞，然后将其处理在易于使用的敏感和高通量细胞排序的仪器上。通过将检测和富集卸载到此类机器上，研究人员所要求的就是将分子封装在薄油壳中的简单工具。高灵敏度和富集提供的应用很多，从罕见的病原体或细胞检测到目标捕获不常见的突变体序列，其目标是下游测序的目标。在降低的成本和功能的扩展之间，这种方法有可能成为一种广泛采用的方法。该SBIR I期项目提议通过与现有流式细胞仪（FC）接口来降低高通量数字PCR（DPCR）。主要的研究目标集中于表征和优化DPCR液滴，用于最初用于细胞分类的多种FC仪器。首先，将优化DPCR液滴的生物学，化学，机械和光学性能，以确保与不同的FC系统的兼容性，同时最大程度地提高早期的PCR效率和特异性。接下来，将运行目标序列的串行稀释液以测试和改善检测阈值。这些样品也将针对当前市售的DPCR系统进行基准测试。同时，将使用FC液滴系统用于研究协作中，以定量和表征感染细胞种群中的HIV潜伏期。 FC处理的液滴的初始结果很有希望，因为由于必须能够接受的细胞类型范围，FC系统已经非常灵活。