Highly Sensitive and Multiplexed Detection of Low-Frequency Mutation in Fragmented ctDNA by a Dual-Role Mediator Blocker Amplification Strategy

Abstract

The detection of mutations in circulating tumor DNA (ctDNA) is challenging due to the significant fragmentation of ctDNA and the high prevalence of the wild-type template. Additionally, variant detection through qPCR is typically dependent on target-specific fluorescence probes, and no more than five targets can be identified in a single reaction due to the limited fluorescence colors in thermal cyclers. To address these limitations, we introduce the Dual-Role Mediator Blocker Amplification (DMBA) strategy, enabling sensitive and multiplex mutation detection without reliance on specific fluorescence probes. This strategy is applicable in both qPCR and melting curve analysis (MCA) platforms. The mediator blockers in DMBA play dual roles: enhancing discrimination between wild-type and mutant DNA and releasing mediator primers. These mediator primers extend the helper target and cleave universal fluorescence probes in qPCR, enabling the detection of mutations at variant allele fractions (VAFs) as low as 0.01%. The DMBA MCA method can identify multiple mutations, overcoming limitations in fluorescence channels by using mediator primers to extend universal fluorescence probes, producing fluorescent double strands with different T_m’s and colors. Multiplexed DMBA-MCA was developed to detect seven variants at 0.1–0.5% VAF in one tube. Our innovative method offers advantages including exceptional sensitivity, elimination of the requirement for specific fluorescence probes, shorter amplicons, and high multiplexing capacity, potentially revolutionizing clinical practice and precision medicine.