Flowthrough thermal regulation enables ultrafast real-time PCR

Although real-time quantitative polymerase chain reaction (real-time qPCR) is the gold standard for molecular diagnostics, prolonged thermal cycling constrains its rapid application. Here, we report an ultrafast real-time qPCR system utilizing dynamic water-flow thermal regulation. By integrating a flexible thin-film carrier with dynamic water flow, the system minimizes interfacial thermal resistance. Additionally, a feedforward predictive control algorithm enables precise temperature control using low-cost solenoid valves. Consequently, the system completes 45 cycles in under 10 min, with heating and cooling ramps of ∼3.7 and ∼0.7 s, respectively. In Mycoplasma pneumoniae validation, the device exhibits superior performance, achieving 10 copies/μL sensitivity, 96.8% efficiency, and R 2 > 0.99 linearity. This work integrates speed, precision, and low cost, demonstrating significant promise for point-of-care diagnostics and large-scale genetic screening.