Breaking through PCR multiplexing barriers: A shared-label-tagged primer design enables high-efficiency...

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Multiplex PCR is crucial for diagnostics but faces challenges like primer-dimer artifacts and nonspecific amplification.
• These limitations restrict throughput and reliability in clinical applications requiring multi-target detection.

Purpose of the Study:

• To introduce a novel shared-label-tagged primer-mediated booster PCR (SLP-bPCR) system.
• To address fundamental limitations of conventional multiplex PCR and enhance detection performance.

Main Methods:

• Development of a unique primer architecture for single-tube nested amplification.
• Integration of SLP-bPCR with droplet digital PCR (ddPCR) for pathogen detection and genotyping.

Main Results:

• SLP-bPCR improved signal-to-background ratio by 1.95-fold and analytical sensitivity by 3.25-fold compared to conventional PCR.
• Achieved reliable 5-plex and 10-plex detection of human genes at low DNA inputs with high efficiency and consistency.
• Demonstrated sensitive and specific identification of bloodborne pathogens and accurate HBV genotyping via ddPCR integration.

Conclusions:

• SLP-bPCR offers a versatile, efficient, and scalable framework for multiplex nucleic acid detection.
• The technology significantly advances high-throughput research and clinical diagnostics, especially for ultra-sensitive multi-analyte profiling.