Multiplexed Quantification of Nucleic Acids with Large Dynamic Range Using Multivolume Digital RT-PCR on a Rotational SlipChip Tested with HIV and Hepatitis C Viral Load

Abstract

In this paper, we are working toward a problem of great importance to global health: determination of viral HIV and Hepatitis C (HCV) loads under point-of-care and resource limited settings. While antiretroviral treatments are becoming widely available, viral load must be evaluated at regular intervals to prevent the spread of drug resistance, and requires a quantitative measurement of RNA concentration over a wide dynamic range (from 50 up to 106 molecules/mL for HIV and up to 108 molecules/mL for HCV). “Digital” single molecule measurements are attractive for quantification, but the dynamic range of such systems is typically limited or requires excessive numbers of wells. Here we designed and tested two microfluidic rotational SlipChips to perform multivolume digital RT-PCR (MV digital RT-PCR) experiments with large and tunable dynamic range. These designs were characterized using synthetic control RNA and validated with HIV viral RNA and HCV control viral RNA. The first design contained 160 wells of each of four volumes (125 nL, 25 nL, 5 nL, and 1 nL) to achieve a dynamic range of 5.2×102 – 4.0×106 molecules/mL at 3-fold resolution. The second design tested the flexibility of this approach, and further expanded it to allow for multiplexing while maintaining a large dynamic range by adding additional wells with volumes of 0.2 nL and 625 nL, and dividing the SlipChip into five regions to analyze five samples each at a dynamic range of 1.8×103 – 1.2×107 molecules/mL at 3-fold resolution. No evidence of cross-contamination was observed. The multiplexed SlipChip can be used to analyze a single sample at dynamic range of 1.7×102 – 2.0×107 molecules/mL at 3-fold resolution with limit of detection of 40 molecules/mL. HIV viral RNA purified from clinical samples were tested on the SlipChip, and viral load results were self-consistent and in good agreement with results determined using the Roche COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test. With further validation, this SlipChip should become useful to precisely quantify viral HIV and HCV RNA for high-performance diagnostics in resource-limited settings. These microfluidic designs should also be valuable for other diagnostic and research applications, including detecting rare cells and rare mutations, prenatal diagnostics, monitoring residual disease, and quantifying copy number variation and gene expression patterns. The theory for the design and analysis of multivolume digital PCR experiments is presented in an accompanying paper by Kreutz et al (Anal. Chem. 2011).

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.…”

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

“…On the other hand, smaller droplets allow the isolation of more molecules per volume assayed. If template concentration is highly variable across samples, both small and large droplet reactions can be carried out in parallel to expand the assay dynamic range 39 .…”

Applications of Digital PCR for Clinical Microbiology

“…Many of these disadvantages are due to technical limitations that are being addressed by manufacturers who are developing improved commercial instruments and reagents (22,23).…”