Biomedical Business Center, Healthcare Business Group , Ricoh Co., Ltd. , 3-25-22 Tonomachi LIC 322, Kawasaki , Kanagawa 210-0821 , Japan.
Division of Analytical Science, Food Research Institute , National Agriculture and Food Research Organization , 2-1-12 Kannondai , Tsukuba , Ibaraki 305-8642 , Japan.
Anal Chem. 2019 Oct 15;91(20):12733-12740. doi: 10.1021/acs.analchem.9b02207. Epub 2019 Sep 23.
Nucleic acid amplification methods, such as polymerase chain reaction (PCR), are extensively used in many applications to detect target DNA because of their high sensitivity, good reproducibility, and wide dynamic range of quantification. However, analytical quality control when detecting low copy number target DNA is often missing because of a lack of appropriate reference materials. Recent advances in analytical sciences require a method to accurately quantify DNA at the single molecule level. Herein, we have developed a novel method to produce reference material containing a defined copy number of target DNA (referred to as "cell number-based DNA reference material"). In this method, a suspension of cells carrying a single target DNA sequence was ejected by an inkjet head, and the number of cells in each droplet was counted using highly sensitive cameras. The resulting solutions contained a defined copy number of target DNA and could be used as reference materials. The use of the newly developed reference material was compared with that of diluted solutions of target DNA to evaluate the performance of qualitative real-time PCR in terms of the limit of detection (LOD). Our results demonstrated that cell number-based DNA reference material provides more accurate information regarding performance quality. The reference material produced by this method is a promising tool to evaluate assay performance.
核酸扩增方法，如聚合酶链反应（PCR），由于其高灵敏度、良好的重现性和广泛的定量动态范围，被广泛应用于许多应用中以检测目标 DNA。然而，由于缺乏适当的参考材料，检测低拷贝数目标 DNA 的分析质量控制往往缺失。分析科学的最新进展需要一种能够在单分子水平上准确定量 DNA 的方法。在此，我们开发了一种生产含有特定目标 DNA 拷贝数的参考材料的新方法（称为“基于细胞数的 DNA 参考材料”）。在该方法中，通过喷墨头喷射携带单个目标 DNA 序列的细胞悬浮液，并使用高灵敏度相机对每个液滴中的细胞数量进行计数。得到的溶液含有特定拷贝数的目标 DNA，可作为参考材料使用。将新开发的参考材料与目标 DNA 的稀释溶液进行比较，以评估定性实时 PCR 在检测限 (LOD) 方面的性能。我们的结果表明，基于细胞数的 DNA 参考材料提供了更准确的性能质量信息。该方法制备的参考材料是评估分析性能的有前途的工具。
