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Generic Reporter Sets for Colorimetric Multiplex dPCR Demonstrated with 6-Plex SNP Quantification Panels.
Maximilian Neugebauer1,2, Silvia Calabrese1, Sarah Müller1
1Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
International Journal of Molecular Sciences
|August 29, 2024
Summary
We developed a generic reporter set for digital PCR (dPCR) to simplify multiplex assay design. This approach decouples target detection from signal generation, enabling optimized fluorescence for new panels and sensitive SNP quantification.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- Digital PCR (dPCR) offers high sensitivity and precision for nucleic acid quantification.
- Designing multiplex dPCR assays with target-specific probes is complex and requires extensive optimization.
- Optimizing fluorescent signals for each new target panel is a significant bottleneck in dPCR assay development.
Purpose of the Study:
- To establish a generic fluorogenic reporter set for multiplex digital PCR to streamline assay development.
- To decouple target detection from signal generation, providing pre-optimized fluorescence signals.
- To demonstrate the utility of generic reporters for sensitive quantification of single-nucleotide polymorphisms (SNPs) in liquid biopsy.
Main Methods:
- Development of a generic 6-plex fluorogenic reporter set utilizing mediator probe technology.
- Tailored selection of fluorophores and quenchers for optimal signal separation.
- Application of the reporter set for multiplex quantification of *KRAS*, *NRAS*, and *BRAF* SNPs using digital PCR.
- Demonstration of designing and optimizing novel generic reporter sets from scratch.
Main Results:
- The generic reporter set demonstrated high population separability in a 6-plex assay.
- Sensitive quantification of SNP targets down to 0.4 copies/µL (10 copies/reaction) was achieved.
- Detection of variant allele frequencies as low as 0.1% against a wild-type background of 400 copies/µL was successful.
- Successful quantification of *KRAS*, *NRAS*, and *BRAF* SNPs, common in liquid biopsy.
Conclusions:
- A generic reporter set simplifies and accelerates the development of multiplex digital PCR assays.
- This technology enables sensitive and precise quantification of low-frequency genetic variants.
- The generic reporter system is adaptable to various digital PCR platforms and multiplexing levels.
- Provides a systematic approach for creating and optimizing novel generic reporter sets.
