# Physical Simulation-Based Calibration for Quantitative Real-Time PCR
## 1 Introduction
The standard curve produced by qPCR is a well-established method for the quantification of nucleic acids [22], in the absence of a gold standard test, the standard curve method is often used to construct and verify the reliability of qPCR instruments for nucleic acid detection [23; 24]. Moreover, following the COVID-19 pandemic, the importance of Ct values has been further emphasized, with many studies focusing on exploring the correlation between Ct values and disease severity and transmissibility [25; 26], as well as the correlation between Ct values and epidemiological trends [21]. In addition to the content of the tested samples, Ct values are also influenced by factors such as the temperature and optical performance of the qPCR instrument itself [27], the use of reagent kits, sample collection time, primer specificity, amplification efficiency, sample inactivation temperature, and inactivation time [28]. To ensure experimental accuracy, researchers can periodically calibrate specific fluorescence channels of qPCR instruments using fluorescence dyes according to the calibration methods provided by the manufacturer [29]. Additionally, qPCR instrument manufacturers generally recommend using the manufacturer-provided matching ROI calibration plates to calibrate the fluorescence signals. This method allows for the collection of the position and intensity of fluorescence signals in each reaction well, thereby determining whether overexposure or underexposure occurs during detection and subsequent correction. However, this step is typically only used when there are noticeable inaccuracies in instrument performance. Users are more inclined to comprehensively confirm the performance of qPCR instruments using positive controls, negative controls, gradient dilution quality control samples, or comparing the results with those obtained from reference instruments [30]. In general, fluorescence detection reagents are sourced from the instrument manufacturer or third-party testing institutions. During the reagent formulation process, strict control of the proportions of each substance is necessary, however, human reading errors are unavoidable. For example, when preparing standard substances of different concentrations through gradient dilution of the same batch of reagents, minor errors in pipetting may lead to the reagent standard substance concentration being lower than the expected target concentration, and this error will be continuously introduced into the entire amplification process [12]. Additionally, properly prepared chemical reagents require strict transportation and storage conditions. However, there are many uncontrollable objective errors during transportation to the laboratory being tested which can result in inaccuracies in chemical reagent detection. Moreover, conventional laboratory quality control and calibration standards still predominantly rely on using standard substances to calibrate the sample linearity of the standard curve of qPCR instruments. The calibration indicators of physical devices differ significantly from those of conventional methods, making them difficult to compare and substitute. In consideration of the recent calibration requirements for PCR instruments, this study proposed a method for temperature-optical calibration of qPCR instruments using a physical method and developed a calibration device based on physical simulation with integrated temperature-fluorescence probes. The calibration device was a PCR amplification simulator that incorporates temperature and standard light sources to synchronize the calibration of the temperature and optical parameters of the PCR instrument. It could calibrate temperature deviation, temperature uniformity, heating and cooling rate, Ct value, melting temperature and other parameters.