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

Area of Science:

• Molecular Biology
• Diagnostics
• Biotechnology

Background:

• Conventional quantitative PCR (qPCR) is standard but slow for time-sensitive diagnostics.
• Existing ultrafast qPCR methods require specialized instruments, limiting clinical use.
• A need exists for ultrafast qPCR on standard microfluidic chips with preserved performance.

Purpose of the Study:

• To develop an ultrafast quantitative PCR (qPCR) method using commercially available microfluidic chips.
• To maintain high sensitivity and specificity for low-copy targets in rapid molecular diagnostics.
• To enable broad clinical implementation without specialized equipment.

Main Methods:

• Established Real-time On-Chip quantitative PCR (ROC-qPCR) using a commercial polymer microfluidic chip.
• Optimized thermal cycling for rapid enzyme activation and denaturation/annealing steps.
• Integrated on-chip fluorescence detection and real-time cycle-to-time conversion.

Main Results:

• Detected African swine fever virus at 10 copies/reaction in approximately 11 minutes with linear quantification.
• Achieved detection of >10 copies within 10 minutes.
• Demonstrated sequence-specific detection without false positives, confirming specificity.

Conclusions:

• ROC-qPCR offers rapid molecular diagnostics (<11 min) without custom instrumentation.
• The method balances speed, sensitivity (10 copies/reaction), and specificity for clinical applications.
• ROC-qPCR is suitable for point-of-care, outbreak response, and decentralized diagnostics.