Kim Jeonghun, Yoo Changhoon, Moon Je Hun, Um Soong Ho
Progeneer Incorporation, 12, Digital-ro 31-gil, Guro-gu, Seoul, 08380, South Korea.
School of Chemical Engineering, Sungkyunkwan University, 2066 Seobu-ro, Suwon, Gyeonggi-do, 16419, South Korea.
Chembiochem. 2020 Sep 1;21(17):2533-2539. doi: 10.1002/cbic.202000179. Epub 2020 May 28.
As the market for personalized lung cancer medicine expands, the demand for molecular diagnostic tools in general, and methods of detecting multiple genes with qualitative, quantitative, and high specificity in particular, have grown. Here, we propose a system for the effective detection of lung cancer-specific, long-length epidermal growth factor receptor (EGFR) gene mutations by using a topological transformation nano-barcoding technique (TNT). In former TNT studies, EGFR was successfully detected in cell environments and at test stages in the presence of a reference gene. However, because typical EGFR target concentrations are significantly lower at the clinical stage and the probe-binding ability of long-length targets is lower that of short targets, our system employs polymerase chain reaction (PCR) amplification, restriction, and filtering (PRF) for EGFR fragmentation to maximize performance. In a PRF system, the target is amplified by PCR, cut to a suitable size by a restriction enzyme, and filtered by a magnetic bead. With detection limits of 0.3555 % and 1.500 % for EGFR Del 19 and L858R mutations, respectively, the proposed TNT with PRF can effectively distinguish mutant cell lines and efficiently detect various lengths of genetic variations in clinical trials.
随着个性化肺癌药物市场的扩大，对分子诊断工具的总体需求，尤其是对能够定性、定量且高特异性地检测多个基因的方法的需求不断增长。在此，我们提出一种利用拓扑转换纳米条形码技术（TNT）有效检测肺癌特异性长片段表皮生长因子受体（EGFR）基因突变的系统。在以往的TNT研究中，在细胞环境以及存在参考基因的测试阶段成功检测到了EGFR。然而，由于临床阶段典型的EGFR靶标浓度显著较低，且长片段靶标的探针结合能力低于短片段靶标，我们的系统采用聚合酶链反应（PCR）扩增、限制性内切酶切割和过滤（PRF）对EGFR进行片段化处理，以实现性能最大化。在PRF系统中，靶标通过PCR扩增，用限制性内切酶切割成合适大小，并用磁珠进行过滤。所提出的带有PRF的TNT对EGFR Del 19和L858R突变的检测限分别为0.3555%和1.500%，在临床试验中能够有效区分突变细胞系，并高效检测各种长度的基因变异。
