综合参考标样法和定标校正法的思想,提出了一种用于校正微流控实时荧光聚合酶链式反应(PCR)系统荧光成像非均匀性的"多目标像素区域定标线性校正"算法, 以提高其检测结果的准确性。以与PCR荧光标记物SYBR Green光谱特性相似的荧光素钠溶液为样本, 检测了11种不同浓度的均匀荧光素钠溶液受激发射荧光信号的强度, 分析了各个目标像素区域荧光强度和荧光素钠溶液浓度之间的线性响应关系, 采用两点定标校正方法计算了CCD各个目标像素区域的校正系数矩阵。实验表明, 3种浓度荧光素钠溶液的成像均匀度分别从校正前的71.28%、72.01%、70.73%提高到校正后的77.49%、80.07%、90.64%; 微流控四腔芯片中相同浓度的DNA样品在PCR扩增阶段的Ct值相对标准偏差由校正前的4.38%、1.94%、3.31%减小到校正后的2.44%、0.79%、1.31%, 显著提高了微流控实时荧光PCR检测结果的准确性。

In combination of reference standard sampling and calibration methods, a multi-target area calibration linear correction algorithm was proposed to correct the nonuniformity of fluorescence detection of microfluidic Polymerase Chain Reaction(PCR) system and to improve the performance of the system. The fluorescein sodium solution with similar spectral characteristics to PCR fluorescence marker SYBR Green was used as the test sample, and the emission fluorescence intensities of eleven uniformity sodium fluorescein solutions with different concentrations were measured. The linear relationship between fluorescence intensities and fluorescein sodium concentrations was analyzed and the correction coefficient matrix in each imaging target area on a CCD was calculated by a two-point linear correction algorithm. The results after correction show that the imaging uniformities in three different concentrations of sodium fluorescein solutions are improved respectively from 71.28%, 72.01%, 70.73% to 77.49%, 80.07%, 90.64%. The relative standard deviations of Ct value for the same concentration DNA template are reduced respectively from 4.38%, 1.94%, 3.31% to 2.44%, 0.79%, 1.31%. These results indicate that the proposed nonuniformity correction algorithm significantly improves the accuracy of microfluidic real-time PCR.