Physical Simulation-Based Calibration for Quantitative Real-Time PCR

The fluorescence quantitative polymerase chain reaction (qPCR) instrument has been widely used in molecular biology applications, where the reliability of the qPCR performance directly affects the accuracy of its detection results. In this paper, an integrated, physics-based calibration device was developed to improve the accuracy and reliability of qPCR, realizing the calibration of qPCR instruments' standard curve through physical simulations. With this calibration device, the collected temperature was used as the control signal to alter the fluorescence output, which allowed different probes to simulate the Ct values corresponding to samples with varying initial concentrations. The temperature and optical performance of this calibration device were tested, followed by a comparative analysis comparing the on-machine test results with standard substances to assess the linearity and uniformity of the Ct values of the measured qPCR instrument. It has been proven that this physical calibration device can effectively replace the biochemical standard substance to carry out comprehensive calibration of the temperature and optical parameters of the qPCR instrument and provide a more reliable method for the periodic calibration and quality control of the qPCR instrument. This contributes to the accuracy and reliability of fluorescence qPCR instruments in the field of molecular biology.