Analysis of Colorectal Cancer Gene Mutations and Application of Long Blocker Displacement Amplification Technology for High-Throughput Mutation Detection.

Abstract

Genetic mutation detection for colorectal cancer (CRC) is crucial for precision diagnosis and treatment, yet current methods often suffer from challenges such as low sensitivity, time consumption, and high costs. In our preliminary bioinformatic analysis of 751 CRC cases from The Cancer Genome Atlas and 131 Chinese patient samples, APC, TP53, and KRAS were identified as the most frequently mutated genes. Among them, KRAS missense mutations emerged as key diagnostic biomarkers. In this study, we applied a fluorescence-based long block displacement amplification (LBDA) sensing method for the rapid, high-throughput, and cost-effective detection of KRAS genetic mutations. In the LBDA system, SYBR Green dye binds to the amplified double-stranded DNA, generating a fluorescence signal that directly reflects the abundance of mutant types (MTs). This real-time signal output enables the enrichment and sensitive detection of MTs, establishing LBDA as an efficient biosensing platform for KRAS genotyping. Using this technique, a detection limit of 0.08% variant allele frequency was achieved with 20 ng of synthetic DNA input. To evaluate clinical performance, the LBDA method was applied to 118 tissue samples from 59 CRC patients, including tumor and matched peritumoral tissues. For 59 CRC tumor samples, LBDA successfully identified KRAS mutations in 37.29% of cases, closely matching results (42.37%) obtained by next-generation sequencing and achieving 88% sensitivity and 100% specificity. In conclusion, this study presents a rapid and cost-effective mutation detection method based on optical biosensing, offering strong potential for advancing personalized CRC diagnosis and treatment.