Theoretical Design and Analysis of Multivolume Digital Assays with Wide Dynamic Range Validated Experimentally with Microfluidic Digital PCR

Abstract

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.

Comparison of Droplet Digital PCR to Real-Time PCR for Quantitative Detection of Cytomegalovirus

“…Few authors, however, have yet investigated the application of this technology to infectious disease diagnostics. Experimental systems have demonstrated proof of principle for quantification of adenoviral genome copies, GB virus in transfected cell culture lines, serial dilutions of hepatitis C virus and HIV RNA, and purified, serially diluted HIV RNA from two clinical samples (19,30,31). The latter studies demonstrated a 4-log 10 -unit linear range and variable agreement with real-time PCR methods.…”

“…Droplet digital PCR (ddPCR) is such a direct method (17)(18)(19)(20)(21)(22). It relies on limiting partition of the PCR volume, such that a positive result in any of a large number of microreactions (in this case, 20,000) indicates the presence of a single target molecule in a given reaction.…”

Programmable active droplet generation enabled by integrated pneumatic micropumps

“…Moreover, it was found that MV-dPCR offers a wider dynamic range and better sensitivity than single-volume dPCR assays. 44,45 Fig . 5A shows a representative fluorescence image for the end-point dPCR detection of a l-DNA sample using a mixture of four differently sized droplets.…”

The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

“…Gregory L. Shipley, 11 Jo Vandesompele, 6 Carl T. Wittwer, 12 and Stephen A. Bustin 13 There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants.…”