Novel interlock probe-driven ligation-mediated PCR platform for ultra-specific detection of low-frequency single-nucleotide variation.

Accurate detection of single-nucleotide variations (SNVs) in circulating tumor DNA (ctDNA) is crucial for early cancer diagnosis and personalized therapy. Conventional methods face challenges owing to low variant allele frequencies and high sequence homology with wild-type (WT) DNA. In the present study, we developed an interlock probe-based ligation-mediated PCR (IP-LMPCR) system that imposes stringent structural constraints to ensure that ligation occurs only upon perfect hybridization. This innovative approach effectively reduces false-positive signals from WT sequences and significantly enhances specificity. The system achieves sub-picomolar sensitivity, with a detection limit of 0.32 pM for KRAS G12D, and can identify mutations at 0.1 % abundance in complex mixtures. Moreover, the interlock probes exhibited exceptional specificity for discriminating single-base mutations even in the presence of homologous WT sequences. Clinical validation using blood samples from colorectal cancer patients confirmed the accuracy and reliability of the method. This user-friendly platform provides a sensitive, specific, and high-confidence approach for detecting SNVs in ctDNAs, exhibiting considerable potential for cancer monitoring and precision medicine. By minimizing off-target ligation, improving the compatibility with fragmented DNA, and achieving single-nucleotide resolution, this technology has established a new standard for liquid biopsy applications in precision oncology.