A Self-Priming Digital Polymerase Chain Reaction Chip for Multiplex Genetic Analysis

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Bioengineering

Background:

• Digital PCR (polymerase chain reaction) is essential for nucleic acid quantification.
• Current multiplexing in digital PCR is limited by cost and complexity.
• Existing methods face challenges with reagent competition and sample handling.

Purpose of the Study:

• To develop a cost-effective digital PCR chip with enhanced multiplex detection capabilities.
• To overcome limitations in current digital PCR multiplexing strategies.
• To enable rapid and precise quantification of nucleic acids using low-cost instrumentation.

Main Methods:

• Designed and tested a self-priming digital PCR chip with six detection areas and four-layer structures.
• Utilized self-priming to preintroduce specific reaction mixes, avoiding primer pair competition.
• Employed monochrome fluorescence for 6-plex detection.

Main Results:

• Achieved multiplex digital detection in a shorter time with reduced reagent consumption.
• Demonstrated precise quantification of five EGFR gene mutations in 15 lung cancer patient blood samples.
• Validated the chip's performance for high-performance diagnostics and liquid biopsies.

Conclusions:

• The self-priming digital PCR chip offers a simple, inexpensive solution for multiplex nucleic acid quantification.
• This technology is suitable for high-performance diagnostics, including liquid biopsies, prenatal diagnostics, and pathogen detection.
• The developed chip addresses key limitations in digital PCR multiplexing, paving the way for broader applications.