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.

这个小企业创新研究（SBIR）项目的更广泛的影响/商业潜力将是能够从大量的背景人群中检测和富集罕见的DNA或RNA分子。目前，这种检测是在昂贵的专用仪器上进行的，这些仪器灵敏度低，不能富集。所提出的策略是将荧光识别的靶分子封装在薄油壳中，使它们模拟细胞，然后在容易获得的灵敏和高通量细胞分选仪器上处理它们。通过将检测和浓缩工作转移到这样的机器上，研究人员所需要的只是一个简单的工具，将分子封装在薄的油壳中。由高灵敏度和富集提供的应用是众多的，从稀有病原体或细胞检测到以下游测序为目标的不常见突变序列的靶向捕获。在降低成本和扩大能力之间，这种方法有可能成为一种被广泛采用的方法。SBIR第一阶段项目提出通过与现有流式细胞仪（FC）接口，以大幅降低成本的方式实现高通量数字PCR（dPCR）。主要研究目标集中在表征和优化dPCR液滴，以用于最初用于细胞分选的各种FC仪器。首先，将优化dPCR液滴的生物、化学、机械和光学性质，以保证与不同FC系统的相容性，同时最大化早期PCR效率和特异性。接下来，将运行靶序列的系列稀释液以测试和提高检测阈值。这些相同的样品也将根据目前市售的dPCR系统进行基准测试。与此同时，FC上的液滴系统将用于研究合作，以定量和表征感染细胞群中的HIV潜伏期。FC处理液滴的初步结果是有希望的，因为FC系统已经非常灵活，因为它们必须能够接受的细胞类型的范围。