Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, United States.
J Am Chem Soc. 2011 Nov 9;133(44):17705-12. doi: 10.1021/ja2060116. Epub 2011 Oct 13.
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.
在即时护理和资源有限的环境下测定病毒 HIV 和丙型肝炎（HCV）载量。虽然抗逆转录病毒疗法已广泛应用，但为了防止耐药性的传播，必须定期评估病毒载量，这需要对 RNA 浓度进行定量测量，测量范围很宽（HIV 为 50 到 10^6 分子/ml，HCV 为 10^8 分子/ml）。“数字”单分子测量在定量方面很有吸引力，但此类系统的动态范围通常有限，或者需要大量的隔室。在这里，我们设计并测试了两个微流旋转 SlipChip 来进行多体积数字 RT-PCR（MV 数字 RT-PCR）实验，具有较大的和可调节的动态范围。这些设计使用合成对照 RNA 进行了表征，并使用 HIV 病毒 RNA 和 HCV 对照病毒 RNA 进行了验证。第一个设计包含 160 个四个体积（125 nL、25 nL、5 nL 和 1 nL）的每个体积的 160 个孔，以实现 5.2×10^2 到 4.0×10^6 分子/ml 的 3 倍分辨率的动态范围。第二个设计测试了这种方法的灵活性，并通过添加体积为 0.2 nL 和 625 nL 的额外孔，以及将 SlipChip 分为五个区域，以在 3 倍分辨率下分析每个样本的五个样本，从而进一步扩展了该方法，同时保持较大的动态范围，从而实现了更大的动态范围。在 5.2×10^2 到 4.0×10^6 分子/ml 的动态范围内，以 3 倍分辨率，每个样本的动态范围为 1.8×10^3 到 1.2×10^7 分子/ml。未观察到交叉污染的证据。带有多路复用的 SlipChip 可以在 3 倍分辨率下对单个样本进行分析，其动态范围为 1.7×10^2 到 2.0×10^7 分子/ml，检测限为 40 分子/ml。从临床样本中提取的 HIV 病毒 RNA 在 SlipChip 上进行了测试，病毒载量结果与使用 Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test 确定的结果一致。经过进一步验证，该 SlipChip 将成为在资源有限的环境中精确定量 HIV 和 HCV RNA 的有力工具，用于高性能诊断。这些微流设计也应该对其他诊断和研究应用有价值，包括检测稀有细胞和稀有突变、产前诊断、监测残留疾病、以及定量拷贝数变异和基因表达模式。Kreutz 等人在其他工作中提出了多体积数字 PCR 实验的设计和分析理论。
