Kim Hanyoup, Ruby Aaron E, Shandilya Harini G, Virmani Arvind K, Rahman Nandita, Strange Christina M, Huuskonen Jarkko
Canon US Life Sciences, Inc., 9800 Medical Center Drive Suite C-120, Rockville, MD 20850, USA.
Biotechniques. 2018 Oct;65(4):205-210. doi: 10.2144/btn-2018-0111.
We have developed a simple and robust probe-free quantitative PCR (qPCR) assay method that can detect minor mutant alleles with a frequency as low as 0.1% in a heterogeneous sample by introducing a novel T-blocker concept to the allele-specific PCR method. Four new KRAS and BRAF mutation detection assays were developed and their performance was demonstrated by testing a large number of replicates, utilizing a customized PCR protocol. Highly efficient and specific mutant amplification in conjunction with selective wild-type suppression by the T-blocker concept enabled 0.1% detection sensitivity using the intercalating dye-based qPCR chemistry instead of more complex target-specific dye-labeled probes. Excellent consistency in sensitivity and specificity of the T-blocker assay concept was demonstrated.
我们开发了一种简单且稳健的无探针定量聚合酶链反应（qPCR）检测方法，通过在等位基因特异性PCR方法中引入新的T阻断剂概念，能够在异质样本中检测频率低至0.1%的次要突变等位基因。我们开发了四种新的KRAS和BRAF突变检测方法，并通过使用定制的PCR方案进行大量重复测试来证明其性能。T阻断剂概念结合高效且特异的突变体扩增以及对野生型的选择性抑制，使用基于嵌入染料的qPCR化学方法实现了0.1%的检测灵敏度，而无需使用更复杂的靶标特异性染料标记探针。结果证明了T阻断剂检测概念在灵敏度和特异性方面具有出色的一致性。
