Improving the Accuracy of Single-Nucleotide Variant Diagnosis Using On-Off Discriminating Primers

Early detection of rare mutations through liquid biopsy can provide real-time information related to cancer diagnosis, prognosis, and treatment outcomes. Cell-free DNA samples used in liquid biopsies contain single-nucleotide variants (SNVs) with a variant allele frequency (VAF) of approximately <= 1%. Droplet digital polymerase chain reaction (ddPCR) is considered the gold standard of sequencing using liquid samples, generating amplicons from samples containing mutations with 0.001-0.005% VAF; however, it requires expensive equipment and time-consuming protocols. Therefore, various PCR methods for discriminating SNVs have been developed; nonetheless, non-specific amplification cannot be avoided even in the absence of mutations, which hampers the accurate diagnosis of SNVs. In this study, we introduce single-nucleotide variant on-off discrimination-PCR (Soo-PCR), a highly accurate and practical method that uses a 3 '-end tailing primer for the on-off discrimination of low-abundance mutant-type targets, including SNVs. Soo-PCR minimizes the chance of incorrect judgments owing to its high discriminating power. Cancer markers, such as KRAS G12D, EGFR L858R, and EGFR T790M mutations, containing 0.1% VAF, were clearly detected in under 2 h with a high reliability comparable with that of ddPCR. This new method serves as a practical approach to accurately detect and evaluate low-abundance mutations in a user-friendly manner.

Automated summary

Early detection of rare mutations through liquid biopsy provides real-time information for cancer diagnosis, prognosis, and treatment outcomes. However, current methods like ddPCR are expensive and time-consuming, and non-specific amplification hampers accurate diagnosis of SNVs. This study introduces Soo-PCR, a practical and highly accurate method for detecting low-abundance mutations, including SNVs, with comparable reliability to ddPCR, but in under 2 hours.