探讨降低实时荧光PCR定量分析系统误差的对策

The exploration to reduce the system error of real-time fluorescent PCR quantitative analysis

目的:探讨降低实时荧光PCR定量分析系统误差的对策。方法:根据现有的和导出的实时荧光PCR的动力学方程和定量分析的线性回归方程,分析和比较基于热循环数或热循环时间描述信号变化的计量精确度及测定靶模板数的系统误差。结果:热循环数的计量精确度为1次,基于热循环数测定靶模板数的系统误差接近于±50.00%。热循环时间的计量精确度至少可以达到0.01 s,基于热循环时间测定靶模板数可在不改变灵敏度的前提下,使定量分析的系统误差接近于±0.02%,比基于热循环数的系统误差低99.96%。结论:在实时荧光PCR中以热循环时间替代热循环数是可行的,其优势是能够大幅度降低实时荧光PCR定量分析的系统误差。

Objective：To explore the countermeasures to reduce the system error of real-time fluorescent PCR quantitative analysis.Methods： According to the dynamic equation of real-time fluorescent PCR and the linear regression equation of quantitative analysis,the measurement accuracy and system error of thermal cycler in the case that the signal changes are described by the number of thermal cycling and the time of thermal cycling is compared and analyzed.Results： When the cycle number acts as measuring unit,the measurement accuracy is 1 time and the system error is close to ±50%;When the cycle time acts as measuring unit,the measurement accuracy is 0.01 second and the system error is close to ±0.02%,it is 99.96% lower than the former.Conclusion： Thermal cycle time replacing cycle number works as measuring unit in real-time fluorescence thermal cycler is feasible.Its advantage is to significantly reduce the system error of real-time fluorescence PCR quantification.