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

这一小型企业创新研究(SBIR)项目的更广泛影响/商业潜力将是能够负担得起从大量背景人群中检测和浓缩稀有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，无法进行浓缩。建议的策略是将荧光识别的目标分子封装在薄的油壳中，使它们模仿细胞，然后在灵敏和高通量的细胞分选仪器上处理它们。通过将检测和浓缩工作转移给这样的机器，研究人员所需要的只是一个简单的工具，将分子封装在薄薄的油壳中。从罕见的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，高灵敏度和富集性提供了大量的应用。在降低成本和扩展能力之间，这种方法有可能成为一种被广泛采用的方法。这个SBIR第一阶段项目建议通过与现有的流式细胞仪(FC)接口，在显著降低成本的情况下实现高通量数字聚合酶链式反应(DPCR)。主要研究目标是表征和优化dPCR液滴，以用于最初用于细胞分选的各种FC仪器。首先，dPCR液滴的生物、化学、机械和光学特性将得到优化，以确保与不同的FC系统兼容，同时最大限度地提高早期的聚合酶链式反应效率和特异性。接下来，将对靶序列进行连续稀释，以测试和提高检测阈值。这些样品也将与目前商业上可用的dPCR系统进行基准比较。同时，水滴-on-FC系统将用于一项研究合作，以量化和表征感染细胞群体中的艾滋病毒潜伏期。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。

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.