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

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 Tm’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.