NGS 是一种识别和确认未知致病菌的前景广阔的技术，然而其在生物防御和公共健康应用等方面的时效性，却往往因为缺乏快速、有效、可靠的自动DNA样品制备方法而受到限制。为了突破这种限制，Kim 等设计了一种基于流体分布元件的数字微流体(DMF) 平台，使得多子系统模块能够进入自动NGS库样品制备系统。通过一种新型毛细管接口，可以实现液体在DMF设备与外部流体模块间的高重复性转移，使得流路连续，且小滴样品能够在一个完整的系统内得到处理。这里，该技术强调了DMF元件平台和NGS 样品制备流程中自动运行毛细管接口的效用。将毛细管接口与一种嵌入式非接触电导检测器连接，DMF设备实现了目标分析物从样品流到小液滴的闭环自动片段采集。NGS 中重复次数最多的缓冲液交换与样品清洗通过使用一种磁珠解决，实现了DMF平台上平均DNA回收率为(80±4.8)%。

先天性糖基化缺陷是由于机体缺少糖基化作用，主要影响到N 相关途径造成的。至少40% 的先天性糖基化缺陷病人在诊断过程中没有得到分子水平的确认。Jones 等通过已经导致先天性糖基化缺陷基因的下一代测序验证的研究，从分子水平提高了诊断先天性糖基化缺陷的水平。12例未知样本致病突变的先天性糖基化缺陷病人作为阳性对照进行NGS验证，分别采用RainDance 与Fluidigm 平台( dPCR) 进行序列富集和目标扩增，SOLiD平台用于测序和扩增产物。进而通过NextGENe 进行生物信息学分析。结果表明，12个阳性对照的致病突变通过NGS都得以确认。在病人诊断过程中，NGS的发展使实验室诊断多基因与分析逐个基因相比成本消耗更低、速度更快、效率更高。临床实验室的下一代测序数据分析结果同样支持这项技术，推广使用这项技术至关重要。

4 总结与展望

生物学的基础研究和分子技术的前进伴随着更精确和更灵敏的测量技术发展。值得一提的是，dPCR具有测量独立性与无需任何校准物的特点。因此，该技术是潜在的核酸测量基准方法，并从原理上为核酸计量提供了保证。相比其他方法，dPCR作为绝对定量方法能准确定量目标DNA和提供可靠的定量数据。商业化dPCR 仪器( 如Fluidigm 公司的BioMark System)的大量出现进一步推动和扩大了该技术的发展和应用范围。

dPCR技术及其应用凸显了单分子定量技术的潜力。不久，微流体dPCR就会突破反应速度和体积的限制，实现自动化和高通量的应用。核酸测序将是基于dPCR的单分子扩增技术最重要的应用领域。dPCR的克隆扩增可以减少下一代测序的时间和成本，并使个人基因组测序得以实现。我们期望在不远的将来，这项技术的发展将对单分子核酸扩增领域产生深远影响，在分子生物学和医学等基础研究和应用方面发挥更大的作用。

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