Fast, sensitive, and specific multiplexed single-molecule detection of circulating tumor DNA

Circulating tumor DNA (ctDNA) analysis has emerged as a highly promising non-invasive assay for detection and monitoring of cancer. However, identification of multiple point-mutant ctDNAs, particularly at extremely low frequencies in early cancer stages, remains a significant challenge. To address this issue, we present a multiplexed ctDNA detection technique, SIMUL (single-molecule detection of multiple low-frequency mutations). SIMUL involves an unbiased preamplification of both wild-type and mutant DNAs, followed by the detection of mutant DNAs through single-molecule multicolor imaging. SIMUL enables highly sensitive and specific detection of multiple single-nucleotide mutations in a short span of time, even in the presence of 10,000-fold excess of wild-type DNA. Importantly, SIMUL can accurately measure mutant fractions due to its linear correlation between the number of single-molecule spots and the variant allele frequency. This breakthrough technique holds immense potential for clinical applications, offering significant improvements for example in early cancer detection and accurate evaluation of anticancer treatment responses.