Precise determination of reaction conditions for accurate quantification in digital PCR by real-time fluorescence monitoring within microwells.

Real-time digital polymerase chain reaction (qdPCR) provides enhanced precision in the field of molecular diagnostics by integrating absolute quantification with process information. However, the optimal reaction conditions are traditionally determined through multiple iterative of experiments. Therefore, we proposed a novel approach to precisely determine the optimal reaction conditions for qdPCR using a standard process, employing real-time fluorescence monitoring within microwells. The temperature-sensitive fluorophore intensity presented the real temperature of each microwell. This enabled us to determine the optimal denaturation and annealing time for qdPCR based on the corresponding critical temperatures derived from the melting curves and amplification efficiency, respectively. To confirm this method, we developed an ultrathin laminated chip (UTL chip) and chose a target that need to be absolutely quantitative. The UTL chip was designed using a fluid‒solid‒thermal coupling simulation model and exhibited a faster thermal response than a commercial dPCR chip. By leveraging our precise determination of reaction conditions and utilizing the UTL chip, 40 cycles of amplification were achieved within 18 min. This was accomplished by precisely controlling the denaturation temperature at 2 s and the annealing temperature at 10 s. Furthermore, the absolutely quantitative of DNA showed good correlation (R > 0.999) with the concentration gradient detection using the optimal reaction conditions with the UTL chip for qdPCR. Our proposed method can significantly improve the accuracy and efficiency of determining qdPCR conditions, which holds great promise for application in molecular diagnostics.