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

Multiplex nucleic acid detection has emerged as an indispensable tool in modern diagnostics, with multiplex PCR established as the gold standard due to its high throughput, cost-effectiveness, and rapid turnaround time. Despite its advantages, conventional multiplex PCR remains constrained by persistent technical challenges-notably primer-dimer artifacts and nonspecific amplification. These limitations not only impose stringent requirements on primer design and reaction optimization but also restrict analytical throughput, ultimately hindering the technology's full potential in critical clinical applications where reliable multi-target detection is paramount.

Here, we present a shared-label-tagged primer-mediated booster PCR (SLP-bPCR) system featuring an innovative primer architecture that enables efficient single-tube nested amplification using a single primer. This transformative approach achieved substantial improvements in multiplex detection performance by simultaneously addressing multiple fundamental limitations of conventional multiplex PCR. Quantitative comparisons demonstrated that the SLP-bPCR system achieved a 1.95 ± 0.10-fold improvement in signal-to-background ratio and a 3.25-fold enhancement in analytical sensitivity compared with conventional PCR. These advancements enabled reliable 5-plex detection of human reference genes at ultralow template inputs (0.01 ng total DNA) with near-ideal amplification efficiency (101.6 % ± 1.2 %), while maintaining robust performance consistency (97.4 % ± 1.9 %) when scaled to more complex 10-plex assays. Moreover, the method integrated seamlessly with droplet digital PCR (ddPCR) platform, enabling sensitive (100 %), and specific (93.8 %) identification of bloodborne pathogens, alongside precise genotyping of hepatitis B virus (HBV) that showed complete concordance with Sanger sequencing.

The SLP-bPCR technology establishes a transformative framework for multiplex nucleic acid detection, delivering unprecedented analytical performance through its unique combination of versatility, efficiency, and scalability. By addressing critical gaps in throughput, accuracy, and practicality, this strategy is poised to advance both high-throughput research and clinical diagnostics, particularly for applications demanding multi-analyte profiling at extreme sensitivity.