Lee Jina, Kim Ji Hyun, Kang Sun Hyung, Yoo Hee Min
Center for Bioanalysis, Korea Research Institute of Standards and Science (KRISS), Daejeon, Korea.
College of Pharmacy, Chungnam National University, Daejeon, Korea.
J Clin Lab Anal. 2020 Aug;34(8):e23344. doi: 10.1002/jcla.23344. Epub 2020 Apr 24.
In standard analytical conditions, an isolation step is essential for circulating tumor DNA (ctDNA) analysis. The necessity of this step becomes unclear with the development of highly sensitive detection methods. The aim of this study was to evaluate ctDNA mimetic nDNA detection as reference materials (RMs) using dPCR technologies either directly from serum or without serum.
To determine an absolute count of both mutation and wild-type bearing DNA molecules, genomic DNA (gDNA) and nucleosomal DNA (nDNA), which are similar in size to cell-free DNA, were evaluated. We tested 3 KRAS mutations in colorectal cancer cell lines.
We describe the recent progress in RMs. The short DNA fragments, such as sDNA and nDNA, exhibited higher quantitative values of dPCR compared to gDNA. The efficiency between Atlantis dsDNase (AD) and Micrococcal Nuclease (MN) affects DNA quantification. Moreover, there was a significant difference in dPCR output when spiking gDNA or nDNA containing KRAS mutations into FBS compared to the dPCR output under non-FBS conditions.
The matrix effect crucially affects the accuracy of gDNA and nDNA level estimation in the direct detection of mimic of patient samples. The form of reference material we proposed should be optimized for various conditions to develop reference materials that can accurately measure copy number and verify the detection of KRAS mutations in the matrix.
在标准分析条件下，循环肿瘤DNA（ctDNA）分析中分离步骤至关重要。随着高灵敏度检测方法的发展，这一步骤的必要性变得不明确。本研究的目的是使用数字PCR技术，直接从血清或无血清状态下评估ctDNA模拟物基因组DNA（nDNA）检测作为参考物质（RMs）。
为了确定携带突变和野生型的DNA分子、基因组DNA（gDNA）和核小体DNA（nDNA）的绝对计数，对大小与游离DNA相似的这些物质进行了评估。我们在结肠癌细胞系中检测了3种KRAS突变。
我们描述了参考物质方面的最新进展。短DNA片段，如小DNA（sDNA）和nDNA，与gDNA相比，在数字PCR中表现出更高的定量值。亚特兰蒂斯双链DNA酶（AD）和微球菌核酸酶（MN）之间的效率影响DNA定量。此外，与非胎牛血清（FBS）条件下的数字PCR输出相比，将含有KRAS突变的gDNA或nDNA加入FBS时，数字PCR输出存在显著差异。
在直接检测患者样本模拟物时，基质效应严重影响gDNA和nDNA水平估计的准确性。我们提出的参考物质形式应针对各种条件进行优化，以开发能够准确测量拷贝数并验证基质中KRAS突变检测的参考物质。
