Ultrafast and real-time on-chip quantitative polymerase chain reaction (ROC-qPCR) with sequence-specific signaling

Quantitative PCR is the standard method for sensitive and specific nucleic acid detection, but conventional qPCR-based protocols require long thermal cycling times that limit use in time critical diagnostics. Ultrafast qPCR platforms that use nanoplasmonic or photonic heating can complete cycling within minutes, yet they depend on specialized instruments and custom components that limit clinical deployment. There is a need for an ultrafast qPCR strategy that operates on commercially available microfluidic chips with standard reagents and maintains quantitative performance for low-copy targets.

We established Real-time On-Chip quantitative PCR (ROC-qPCR), which combines a commercial polymer-based microfluidic PCR chip with Taq polymerase and TaqMan probes. Thermal cycling was optimized to 40 s of enzyme activation, followed by 50 cycles of 89 °C for 1 s and 68 °C for 1 s, with on-chip fluorescence detection and time conversion from cycle number. Under these conditions, the African swine fever virus p72 gene was detected at 10 copies per reaction in about 11 min with linear quantification (R² = 0.9697) over serial dilutions. Samples containing more than 10 copies were detected within 10 min. No amplification was observed from porcine genomic DNA or non-target controls, and the probe design enabled sequence-specific detection without false positive signals.

ROC-qPCR resolves trade-off between detection speed, accuracy, and specificity in rapid molecular diagnostics while requiring no custom instrumentation. The method shortens detection time to the 10 min range while preserving sensitivity and specificity at 10 copies per reaction. This platform is ready for real-world translation into point-of-care workflows, outbreak response testing, and decentralized molecular diagnostics. The approach shows that clinically reliable ultrafast PCR can be broadly implemented without specialized systems, enabling practical diagnostic scaling.