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

Area of Science:

• Molecular Biology
• Genetics
• Oncology

Background:

• Digital PCR (dPCR) offers high sensitivity for low-frequency variant detection but faces limitations in multiplexing capacity with conventional 2-color systems.
• Simultaneous detection of multiple cancer-related gene mutations is critical, especially for KRAS and GNAS, key drivers in pancreatic cancer development, often found at low levels in clinical samples.

Purpose of the Study:

• To develop and validate advanced 6-color dPCR assays for enhanced multiplexing capabilities in detecting KRAS and GNAS mutations.
• To assess the sensitivity, specificity, and concordance of novel dPCR assays compared to existing methods for clinical application.

Main Methods:

• Two distinct 6-color dPCR assays, PlexScreen-dPCR (screening) and PlexID-dPCR (variant-specific), were developed on a droplet-based platform.
• Customized 6-fluorophore primer/probe sets were utilized, and assays were validated using synthetic DNA, cell lines, tissue, and duodenal fluid samples.

Main Results:

• PlexScreen-dPCR achieved limits of detection (LOD) as low as 0.03%-0.06%, enabling sensitive detection of low-abundance mutations.
• PlexID-dPCR successfully identified all 14 predefined KRAS and GNAS variants in a single reaction with high accuracy.
• Both assays demonstrated complete concordance with conventional methods and strong correlation in variant allele frequency quantification.

Conclusions:

• The developed 6-color dPCR assays provide scalable, high-throughput solutions for detecting KRAS and GNAS mutations, supporting clinical integration.
• Assay compatibility with existing platforms and streamlined workflows facilitate adoption, with potential for further optimization in high-plex and broader genomic applications.