Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois 60637, United States.
Anal Chem. 2011 Nov 1;83(21):8158-68. doi: 10.1021/ac201658s. Epub 2011 Oct 7.
This paper presents a protocol using theoretical methods and free software to design and analyze multivolume digital PCR (MV digital PCR) devices; the theory and software are also applicable to design and analysis of dilution series in digital PCR. MV digital PCR minimizes the total number of wells required for "digital" (single molecule) measurements while maintaining high dynamic range and high resolution. In some examples, multivolume designs with fewer than 200 total wells are predicted to provide dynamic range with 5-fold resolution similar to that of single-volume designs requiring 12,000 wells. Mathematical techniques were utilized and expanded to maximize the information obtained from each experiment and to quantify performance of devices and were experimentally validated using the SlipChip platform. MV digital PCR was demonstrated to perform reliably, and results from wells of different volumes agreed with one another. No artifacts due to different surface-to-volume ratios were observed, and single molecule amplification in volumes ranging from 1 to 125 nL was self-consistent. The device presented here was designed to meet the testing requirements for measuring clinically relevant levels of HIV viral load at the point-of-care (in plasma, <500 molecules/mL to >1,000,000 molecules/mL), and the predicted resolution and dynamic range was experimentally validated using a control sequence of DNA. This approach simplifies digital PCR experiments, saves space, and thus enables multiplexing using separate areas for each sample on one chip, and facilitates the development of new high-performance diagnostic tools for resource-limited applications. The theory and software presented here are general and are applicable to designing and analyzing other digital analytical platforms including digital immunoassays and digital bacterial analysis. It is not limited to SlipChip and could also be useful for the design of systems on platforms including valve-based and droplet-based platforms. In a separate publication by Shen et al. (J. Am. Chem. Soc., 2011, DOI: 10.1021/ja2060116), this approach is used to design and test digital RT-PCR devices for quantifying RNA.
本文提出了一种使用理论方法和免费软件设计和分析多体积数字 PCR（MV 数字 PCR）设备的方案；该理论和软件也可用于设计和分析数字 PCR 中的稀释系列。MV 数字 PCR 在保持高动态范围和高分辨率的同时，最大限度地减少了“数字”（单分子）测量所需的总孔数。在一些示例中，预测具有少于 200 个总孔的多体积设计将提供具有与需要 12000 个孔的单体积设计相似的 5 倍分辨率的动态范围。利用并扩展了数学技术来最大限度地从每个实验中获取信息，并量化设备的性能，并使用 SlipChip 平台进行了实验验证。MV 数字 PCR 可靠地运行，并且来自不同体积的孔的结果彼此一致。没有观察到由于不同的表面积与体积比而产生的伪影，并且在 1 至 125 nL 的体积范围内的单分子扩增是一致的。本文提出的设备旨在满足在护理点测量临床相关 HIV 病毒载量的测试要求（在血浆中，<500 个分子/mL 至 >1000000 个分子/mL），并且使用 DNA 控制序列对预测的分辨率和动态范围进行了实验验证。这种方法简化了数字 PCR 实验，节省了空间，从而能够在一个芯片上的每个样品的单独区域进行多路复用，并促进了用于资源有限应用的新型高性能诊断工具的开发。本文提出的理论和软件是通用的，可用于设计和分析其他数字分析平台，包括数字免疫测定和数字细菌分析。它不仅限于 SlipChip，对于基于阀和基于液滴的平台等平台上的系统设计也可能有用。在 Shen 等人的另一篇出版物中（J. Am. Chem. Soc.，2011，DOI：10.1021/ja2060116），这种方法用于设计和测试用于定量 RNA 的数字 RT-PCR 设备。
