# Physical Simulation-Based Calibration for Quantitative Real-Time PCR
## 5 Discussion
When employing biological methods for preparing standard curves during calibration processes, these curves are influenced not only by inherent instrument performance but also errors stemming from sample preparation procedures [32], properties associated with dilution buffers [42], precision issues related to pipetting accuracy [43], fluctuations within fluorescence baselines causing measurement errors, DNA extraction efficiency [44,45], systematic drifts observed during standard curve measurements [12], as well as environmental impacts related to storage and transportation [46]. Our proposed physical device effectively mitigates these influences. While a result with a standard curve r \(\geq\) 0.980 is generally considered satisfactory when utilizing biological methods for calibrating purposes, our physical method directly simulates an ideal r = 1-standard curve where any measured deviation is solely linked to qPCR instrument performance. Moreover, our calibration device encompasses features enabling simultaneous adjustment analyses involving both thermal parameters alongside composite optical parameters compared against single parameter adjustments, such as those focused solely on addressing heating/cooling rate within qPCR instruments [14,47]. Based on the simulation principles provided in this article, it is evident that the application of this device also includes the calibration of multiple-channel optical parameters and physical methods for analyzing melting curves. The resulting calibration results can provide improved assessment and calibration guidance for the use of qPCR instruments. The system can customize simulated Ct values and standard curves based on the characteristics of the standard materials or samples used by the user, offering greater flexibility compared to selecting fixed standard materials and providing more targeted calibration results. Furthermore, the increasing use of multiplex qPCR instruments for detection has led to a need for multiple fluorescence channels to be utilized simultaneously in order to detect multiple targets and improve detection efficiency. However, this also necessitates the calibration of all fluorescence channels of qPCR instruments [48], typically requiring costly and complex configurations with standard substances containing multiple fluorescent probes. The device developed in this study utilizes LEDs as the standard light source, enabling full-spectrum optical simulation and calibration of different fluorescence channels of the PCR instrument through selection via the device's filters. This approach not only reduces costs but also offers good reproducibility while achieving simultaneous calibration of multiple channels, thereby saving time and improving overall calibration efficiency. The device provides robust assurance for the application of PCR instruments in molecular biology research. Through performance testing and comparative on-site experiments, we have demonstrated that this calibration device can serve as an alternative method with superior capabilities compared to traditional biochemical approaches, indicating its significant potential for future applications in research settings. This study also has a limitation. The threshold cycle and the amplification efficiency are two crucial quantitative parameters [49,50]. Through the fluorescence output control of the calibration device, a simulation of specific Ct values is achieved, and standard curve simulations with a linearity of one are realized. However, according to the amplification efficiency calculation, Equation (6) [13,20] is as follows:  
\[E=10^{-(1/\text{slope})}-1 \tag{6}\]  
The theoretically simulated amplification efficiency of the standard curve is 115%, while the actual amplification efficiency is 111.546%. A slope close to \(-\)3.32 indicates optimal, 100% PCR amplification efficiency [12]. When amplifying real biological nucleic acid samples, the ideal PCR reaction amplification efficiency typically ranges from 90% to 110% [51].