Quantitative Multiplex Digital PCR with Fluorescence-Encoded Nanoreactor Beads

Syndromic PCR panels are becoming a pivotal diagnostic tool in precision medicine. Yet, the design and validation of such test panels is cumbersome, and the integration of new markers typically necessitates complete panel revalidation, a challenge that becomes even more complex for quantitative assays. Here, we introduce a novel multiplex digital PCR strategy utilizing fluorescence-encoded magnetic Nanoreactor Beads (femNRB), enabling cross-reactivity-free detection and absolute quantification of multiple genetic markers within a single sample. These uniquely encoded nanoreactor beads are reversibly conjugated to target-specific primers and probes, allowing the formation of highly adaptable panels composed of multiple PCR assays. As proof of concept, we assembled and validated a 10-assay panel targeting species-specific markers for common nosocomial pathogens. We subsequently expanded this panel with six additional markers for antibiotic resistance and virulence, systematically evaluating both configurations. Remarkably, without further optimization, both panels demonstrated equivalent analytical performance, underscoring the robustness and scalability of this method. This approach operates without complex microfluidic hardware and represents a diagnostic multiplex digital PCR platform that enables seamless assay integration while maintaining uncompromised quantification across variable sample concentrations. By eliminating the need for recalibration, this method substantially simplifies assay expansion, providing a powerful, modular solution for developing and upgrading diagnostic test panels with minimal effort.