Sensitive detection of low-abundance in-frame deletions in EGFR exon 19 using novel wild-type blockers in real-time PCR

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.

Fluorometric Detection of Low-Abundance EGFR Exon 19 Deletion Mutation Using Tandem Gene Amplification

“…To date, several methods including Sanger DNA sequencing, next generation sequencing (NGS), and real-time PCR with TaqMan probe have been considered as a standard method for monitoring EGFR mutations [ 8 , 9 ]. Alternatively, there are various PCR-based modifications using DNA-LNA blocker [ 10 ], universal oligo-quencher [ 11 ], allele-specific probe [ 12 ], and graphene oxide [ 13 ]. Despite several PCR-based modifications have been developed in an attempt to detect ctDNA, they still have limitations such as low sensitivity for detecting low frequency of mutant ctDNA in the plasma.…”