Digital PCR (dPCR) enables sensitive and precise quantification of template nucleic acid without calibration. However, dPCR is not yet in widespread use, probably due to the need for expensive specialized instruments. In this paper, we describe a dPCR system using a simple microfluidic chip and common laboratory tools. The microfluidic chip consists of two parts: a PDMS part with 24,840 × 0.25 nL microwells and a PDMS-coated flat glass plate. Human RNase P gene was adopted as the model template. Commercial products of human genomic DNA and real-time PCR reagents were mixed to make a PCR mixture. The PCR mixture was confined to the microwells by the PDMS degas-driven liquid control technique. The thermal cycling was performed on a common well-type thermal cycler with a minor modification. During the thermal cycling, evaporation of the PCR mixture was prevented with a handmade water holder. In the fluorescence image, bright (positive) microwells and dim (negative) ones were clearly discriminated. The number of the positive microwells was counted using software, and was used for estimation of the template concentration in the sample based on the theory of the Poisson distribution. The estimated concentrations well agreed with the input template concentrations in the range from 1.32 copies/µL to 13 200 copies/µL. The techniques presented in this paper will pave the way for facile dPCR in a broad range of laboratories without the need for expensive instruments.

中文翻译：

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使用简单的 PDMS 微流控芯片和标准实验室设备进行数字 PCR。

数字 PCR (dPCR) 无需校准即可对模板核酸进行灵敏、精确的定量。然而，dPCR 尚未广泛使用，可能是因为需要昂贵的专用仪器。在本文中，我们描述了使用简单的微流控芯片和常见实验室工具的 dPCR 系统。微流控芯片由两部分组成：具有 24,840 × 0.25 nL 微孔的 PDMS 部分和 PDMS 涂层的平板玻璃板。采用人RNase P基因作为模型模板。将人类基因组DNA的商业产品和实时PCR试剂混合以制备PCR混合物。通过 PDMS 脱气驱动的液体控制技术将 PCR 混合物限制在微孔内。热循环是在普通井型热循环仪上进行的，并进行了较小的修改。在热循环过程中，用手工制作的水保持器防止 PCR 混合物蒸发。在荧光图像中，可以清楚地区分明亮（阳性）微孔和暗淡（阴性）微孔。使用软件对阳性微孔的数量进行计数，并根据泊松分布理论用于估计样品中的模板浓度。估计浓度与输入模板浓度非常一致，范围为 1.32 拷贝/μL 至 13 200 拷贝/μL。本文介绍的技术将为在广泛的实验室中轻松进行 dPCR 铺平道路，而无需使用昂贵的仪器。