Huang Jun-Fu, Zeng Dong-Zhu, Duan Guang-Jie, Shi Yan, Deng Guo-Hong, Xia Han, Xu Han-Qing, Zhao Na, Fu Wei-Ling, Huang Qing
Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China.
Department of General Surgery, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China.
PLoS One. 2015 Dec 23;10(12):e0145698. doi: 10.1371/journal.pone.0145698. eCollection 2015.
The high degree of intra-tumor heterogeneity has meant that it is important to develop sensitive and selective assays to detect low-abundance KRAS mutations in metastatic colorectal carcinoma (mCRC) patients. As a major potential source of tumor DNA in the aforementioned genotyping assays, it was necessary to conduct an analysis on both the quality and quantity of DNA extracted from formalin-fixed paraffin-embedded (FFPE). Therefore, four commercial FFPE DNA extraction kits were initially compared with respect to their ability to facilitate extraction of amplifiable DNA. The results showed that TrimGen kits showed the greatest performance in relation to the quality and quantity of extracted FFPE DNA solutions. Using DNA extracted by TrimGen kits as a template for tumor genotyping, a real-time wild-type blocking PCR (WTB-PCR) assay was subsequently developed to detect the aforementioned KRAS mutations in mCRC patients. The results showed that WTB-PCR facilitated the detection of mutated alleles at a ratio of 1:10,000 (i.e. 0.01%) wild-type alleles. When the assay was subsequently used to test 49 mCRC patients, the results showed that the mutation detection levels of the WTB-PCR assay (61.8%; 30/49) were significantly higher than that of traditional PCR (38.8%; 19/49). Following the use of the real-time WTB-PCR assay, the ΔCq method was used to quantitatively analyze the mutation levels associated with KRAS in each FFPE sample. The results showed that the mutant levels ranged from 53.74 to 0.12% in the patients analyzed. In conclusion, the current real-time WTB-PCR is a rapid, simple, and low-cost method that permits the detection of trace amounts of the mutated KRAS gene.
肿瘤内高度的异质性意味着开发灵敏且具选择性的检测方法以检测转移性结直肠癌（mCRC）患者中低丰度KRAS突变很重要。作为上述基因分型检测中肿瘤DNA的主要潜在来源，有必要对从福尔马林固定石蜡包埋（FFPE）样本中提取的DNA的质量和数量进行分析。因此，最初比较了四种商用FFPE DNA提取试剂盒促进可扩增DNA提取的能力。结果显示，TrimGen试剂盒在提取的FFPE DNA溶液的质量和数量方面表现最佳。以TrimGen试剂盒提取的DNA为模板进行肿瘤基因分型，随后开发了一种实时野生型阻断PCR（WTB-PCR）检测方法来检测mCRC患者中的上述KRAS突变。结果表明，WTB-PCR能够以1:10,000（即0.01%）的野生型等位基因比例检测突变等位基因。当该检测方法随后用于检测49例mCRC患者时，结果显示WTB-PCR检测方法的突变检测水平（61.8%；30/49）显著高于传统PCR（38.8%；19/49）。在使用实时WTB-PCR检测方法之后，采用ΔCq法对每个FFPE样本中与KRAS相关的突变水平进行定量分析。结果显示，在所分析的患者中，突变水平范围为53.74%至0.12%。总之，当前的实时WTB-PCR是一种快速、简单且低成本的方法，可用于检测痕量的突变KRAS基因。
