A FEN1-EDCR dual-amplification strategy for ultrasensitive detection of CHCHD10 c.176C>T mutation

A single-base mutation in the CHCHD10 gene can impair thermogenic responses, and inhibit the efficient conversion of white adipose tissue to beige adipose tissue. However, achieving high-fidelity detection of such low-abundance single nucleotide polymorphisms (SNPs) within complex biological matrices remains a critical technological challenge. This study developed a novel method for detecting a single-base mutation in CHCHD1 0 by integrating the specific recognition of flap endonuclease 1 (FEN1) with the cascade amplification of an entropy-driven circuit reaction (EDCR), supplemented with magnetic separation to suppress background interference. In this process, the mutant target (MT) hybridizes with the upstream probe (UP) and a hairpin downstream probe (DP) to form a specific three-base overlapping structure. FEN1 recognizes and cleaves this structure, releasing a cleavage sequence (CS). Following magnetic purification, the CS activates the EDCR, enabling dual-signal amplification. The hairpin DP design effectively conceals the CS, significantly reducing background noise. The method achieves a detection limit of 0.23 pM, discriminates mutation abundances as low as 0.01% in mixed DNA samples, and demonstrates robust performance in complex matrices such as human serum and high-background genomic DNA. This work provides an innovative and reliable methodological tool for the detection of low-abundance point mutations in complex samples.