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- Title
Sensitive detection of low-abundance in-frame deletions in EGFR exon 19 using novel wild-type blockers in real-time PCR.
- Authors
Ren, Xiao-Dong; Liu, Ding-Yuan; Guo, Hai-Qin; Wang, Liu; Zhao, Na; Su, Ning; Wei, Kun; Ren, Sai; Qu, Xue-Mei; Dai, Xiao-Tian; Huang, Qing
- Abstract
Epidermal growth factor receptor (EGFR) mutations are associated with response of tyrosine kinase inhibitors (TKIs) for patients with advanced non-small cell lung cancer (NSCLC). However, the existing methods for detection of samples having rare mutations(i.e. ~0.01%) have limits in terms of specificity, time consumption or cost. In the current study, novel wild-type blocking (WTB) oligonucleotides modified with phosphorothioate or inverted dT at the 5′-termini were designed to precisely detect 11 common deletion mutations in exon 19 of EGFR gene (E19del) using a WTB-PCR assay. And internal competitive leptin amplifications were further applied to enhance the specificity of the WTB-PCR system. Our results showed that WTB-PCR could completely block amplification of wild-type EGFR when 200 ng of DNA was used as template. Furthermore, the current WTB-PCR assay facilitated the detection of E19del mutations with a selectivity of 0.01% and sensitivity as low as a single copy. And, the results showed that the current WTB-PCR system exceeded detection limits afforded by the ARMS-PCR assay. In conclusion, the current WTB-PCR strategy represents a simple and cost-effective method to precisely detect various low-abundance deletion mutations.
- Publication
Scientific Reports, 2019, Vol 9, Issue 1, pN.PAG
- ISSN
2045-2322
- Publication type
Article
- DOI
10.1038/s41598-019-44792-1