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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 10(6) molecules/mL for HIV and up to 10(8) 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 compartments. 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 × 10(2) to 4.0 × 10(6) 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 × 10(3) to 1.2 × 10(7) 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 a dynamic range of 1.7 × 10(2) to 2.0 × 10(7) 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 other work by Kreutz et al.

SUBMITTER: Shen F

PROVIDER: S-EPMC3216675 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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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.

Shen Feng F Sun Bing B Kreutz Jason E JE Davydova Elena K EK Du Wenbin W Reddy Poluru L PL Joseph Loren J LJ Ismagilov Rustem F RF

Journal of the American Chemical Society 20111013 44

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 10(6) molecules/mL for HIV and up to 10(8) molecules/ ...[more]

PMID: 21995644

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Project description:In this paper, digital quantitative detection of nucleic acids was achieved at the single-molecule level by chemical initiation of over one thousand sequence-specific, nanoliter isothermal amplification reactions in parallel. Digital polymerase chain reaction (digital PCR), a method used for quantification of nucleic acids, counts the presence or absence of amplification of individual molecules. However, it still requires temperature cycling, which is undesirable under resource-limited conditions. This makes isothermal methods for nucleic acid amplification, such as recombinase polymerase amplification (RPA), more attractive. A microfluidic digital RPA SlipChip is described here for simultaneous initiation of over one thousand nL-scale RPA reactions by adding a chemical initiator to each reaction compartment with a simple slipping step after instrument-free pipet loading. Two designs of the SlipChip, two-step slipping and one-step slipping, were validated using digital RPA. By using the digital RPA SlipChip, false-positive results from preinitiation of the RPA amplification reaction before incubation were eliminated. End point fluorescence readout was used for "yes or no" digital quantification. The performance of digital RPA in a SlipChip was validated by amplifying and counting single molecules of the target nucleic acid, methicillin-resistant Staphylococcus aureus (MRSA) genomic DNA. The digital RPA on SlipChip was also tolerant to fluctuations of the incubation temperature (37-42 °C), and its performance was comparable to digital PCR on the same SlipChip design. The digital RPA SlipChip provides a simple method to quantify nucleic acids without requiring thermal cycling or kinetic measurements, with potential applications in diagnostics and environmental monitoring under resource-limited settings. The ability to initiate thousands of chemical reactions in parallel on the nanoliter scale using solvent-resistant glass devices is likely to be useful for a broader range of applications.

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Dataset Information

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.

Publications

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.

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