Theoretical design and analysis of multivolume digital assays with wide dynamic range validated experimentally with microfluidic digital PCR.
Theoretical design and analysis of multivolume digital assays with wide dynamic range validated experimentally with microfluidic digital PCR.
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DOI：
10.1021/ac201658s
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发表时间：
2011-11-01
影响因子：
7.4
通讯作者：
Ismagilov, Rustem F.
中科院分区：
文献类型：
--
作者：
Kreutz, Jason E.;Munson, Todd;Huynh, Toan;Shen, Feng;Du, Wenbin;Ismagilov, Rustem F.
本文提出了一种利用理论方法和免费软件设计和分析多引物数字PCR（MV digital PCR）装置的方案，该理论和软件同样适用于数字PCR中稀释系列的设计和分析。MV数字PCR最大限度地减少了“数字”（单分子）测量所需的威尔斯总数，同时保持高动态范围和高分辨率。在一些实例中，预测具有少于200个总威尔斯孔的多体积设计提供具有5倍分辨率的动态范围，其类似于需要12，000个威尔斯孔的单体积设计。利用和扩展数学技术以最大化从每个实验获得的信息并量化器件的性能，并使用SlipChip平台进行实验验证。MV数字PCR被证明是可靠的，并且来自不同体积的威尔斯孔的结果彼此一致。未观察到由于不同表面积与体积比而导致的伪影，并且在1至125 nL的体积范围内的单分子扩增是自洽的。本文介绍的设备旨在满足在护理点测量临床相关HIV病毒载量水平的测试要求（血浆中，<500分子/mL至> 1，000，000分子/mL），并且使用DNA的对照序列对预测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，从而通过在一个芯片上为每个样品使用单独的区域来实现多路复用，并促进了针对资源有限的应用开发新的高性能诊断工具。本文所介绍的理论和软件具有通用性，可用于数字化免疫分析和数字化细菌分析等其他数字化分析平台的设计和分析。它不限于滑动芯片，也可以用于平台上的系统设计，包括基于阀和基于液滴的平台。在Shen等人（JACS 2011）的随附论文中，该方法用于设计和测试用于定量RNA的数字RT-PCT装置。
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 by 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 the accompanying paper by Shen et al. (JACS 2011), this approach is used to design and test digital RT-PCT devices for quantifying RNA.
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