Six-Color Multiplex Digital PCR Assays for Comprehensive Screening and Identification of Multiple Driver Mutations...

Digital polymerase chain reaction (dPCR) is widely recognized for its high sensitivity in detecting low-frequency variants; however, conventional 2-color systems have limited multiplex capacity. Expanding this capability is essential for simultaneous detection of multiple driver mutations in cancer-related genes. KRAS and GNAS are key driver genes in the early development of pancreatic cancer and its precursor lesions, and mutations in these genes are often present at low abundance in clinical samples.

Two 6-color dPCR assays were developed using a droplet-based platform. PlexScreen-dPCR is a multicolored drop-off assay designed to screen for mutations in KRAS codons 12/13 and 61 and GNAS codon 201, without specifying individual variants. PlexID-dPCR employs variant-specific probes to distinguish among 14 predefined KRAS and GNAS mutations in a single reaction. The assays were validated using synthetic DNA, cell lines, 23 tissue samples, and 12 duodenal fluid samples. Customized primer/probe sets with 6 fluorophores were employed in a 6-color droplet dPCR system, and the limits of detection (LOD) were determined.

PlexScreen-dPCR, applied in contrived samples, demonstrated LODs as low as 0.03% to 0.06%, enabling high-sensitivity detection of low-abundance mutations. PlexID-dPCR accurately identified all 14 variants in a single well. Both assays showed complete concordance with conventional methods, exhibiting a strong correlation for variant allele frequency quantification.

These 6-color dPCR assays offer scalable solutions for improved throughput detection of KRAS and GNAS mutations. Their compatibility with commercially available platforms and streamlined workflow support their integration into clinical practice. Further optimization can enhance cluster interpretation in high-plex settings and facilitate expansion toward broader genomic targets.