Establishment of a Method for Detecting BRAF V600E Mutation Using an Epitaxial Probe - Fluorescence Quantitative PCR with the classical four - primer ARMS - PCR Architecture

Abstract This study presents an innovative Epitaxial Probe-Allelic Specific PCR (EPAS‑PCR) for highly sensitive and specific fluorescence detection of the BRAF V600E mutation. By attaching a target‑unrelated extension sequence to the 5′ end of the inner allele‑specific primer and pairing it with a universal primer and a TaqMan probe, the method converts the classical four‑primer Amplification Refractory Mutation System PCR (ARMS‑PCR) into a closed‑tube real‑time qPCR system, eliminating gel electrophoresis. The core innovation decouples and cascades mutation recognition with signal generation: only when the inner primer specifically extends the mutant template does the downstream probe system produce fluorescence. The outer primer F1 contributes pre‑amplification and competitively suppresses wild‑type non‑specific amplification. Using Taq polymerase lacking 3′→5′ exonuclease activity is critical for specificity. The assay exhibits good linearity (R² ≥ 0.99) from 1 × 10⁴ to 1 × 10⁸ copies/μL. In plasmid mixtures, it detects 0.137% mutant fraction at 10⁶ copies/reaction and 0.412% at 10⁴ copies/reaction; in genomic DNA standards, the limit of detection is 0.2%, superior to conventional ARMS‑PCR. Compared with ARMS‑PCR, EPAS‑PCR avoids the sensitivity loss and non‑specificity risks arising from omitting outer primers and introducing extra mismatches. Direct comparison shows that ARMS‑PCR without F1 produces false positives around cycle 40, whereas complete EPAS‑PCR remains signal‑free up to 50 cycles. The EPAS‑PCR method offers advantages such as modular design, straightforward development, and reliable performance. In addition to detecting the BRAF V600E mutation, it is also applicable to the detection of other nucleic acid point mutations, representing a novel strategy with broad application prospects.