# Physical Simulation-Based Calibration for Quantitative Real-Time PCR
## 5 Discussion  
The detection of Ct values by qPCR instruments can be influenced by various factors. Additionally, when calibrating the optical parameters of qPCR instruments using biochemical methods, errors resulting from standard substance freeze-thaw cycles, DNA degradation, and serial dilution gradient processes may lead to significant uncertainty in the final linear measurement results. This paper presents the design and development of a calibration device utilizing traceable standard light sources combined with temperature probes. This device enables the simulation of standard curves solely through physical quantity control, facilitating the calibration of both temperature and optical performance of qPCR instruments. By employing this device to simulate standard Ct values and the standard curve, it becomes possible to better mitigate differences in Ct value detection results introduced by other influencing factors. Specifically, the assessment focuses solely on differences in Ct values resulting from temperature non-uniformity and optical detection design flaws in the qPCR instrument. This paper presents a calibration device based on physical simulation method that incorporates dual temperature and optical calibration functions. This device is capable of real-time acquisition of temperature data from the measured qPCR instruments and can simulate optical amplification. It can be used to simulate uniformity calibration with standard materials of the same concentration and linear calibration with gradient-diluted standard materials for standard curve generation. The potential of this device lies in its ability to address both temperature and optical calibrations, which are typically conducted separately or only focusing on temperature in conventional qPCR instruments performance calibrations [32; 33; 34; 35]. The effects caused by temperature uniformity and fluorescence system noise are often analyzed independently, leading to challenges in verifying differences in temperature uniformity through repeated sample amplification experiments on a 96-well plate [36]. Optical calibration usually relies on biochemical methods for generating standard curves using one or more standard substances [37; 38], or comparing positive and negative amplification results from qPCR experiments performed under identical conditions using two or more PCR instruments [39; 40]. However, due to variations in qPCR assays performed in different laboratories as well as the diverse nature of standard substances used, direct comparison of quantitative data between different labs becomes challenging [41]. Furthermore, biological method-based calibrations yield composite results that make it difficult to discern subtle differences arising from hardware manufacturing variances within the optical system or discrepancies introduced by biological samples preparation procedures and human error affecting amplification outcomes. Regular comprehensive evaluations of qPCR instruments performance are essential for meeting quality control requirements while ensuring accurate and reliable amplification results. In contrast \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Probe Position** & **A7** & **B7** & **C7** & **D7** & **E7** & **F7** & **G7** & **H7** \\ \hline Measured Ct value & 24.18 & 24.33 & 24.22 & 24.45 & 24.57 & 24.67 & 24.78 & 24.82 \\ Average value & & & & & 24.50 & & \\ Uniformity of Ct & & & & 0.64 & & & \\ Precision of Ct & & & & 1.01\% & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 8: The calibration device simulates measured results with identical Ct values. to biological methods, our proposed physical method offers greater stability and reliability in terms of control.