基于主成分分析的多重定量PCR荧光串扰校正

Fluorescence Crosstalk Correction for Multiple Quantitative PCR Based on Principal Component Analysis

聚合酶链式反应(PCR)是分子生物学常用的检测手段,主要用于对生物的DNA或RNA进行检测。由于荧光光谱重叠和滤光片过滤带宽限制,检测时所获得的荧光数据通常会包含荧光通道之间的串扰,串扰的存在使PCR结果分析变得复杂,并可能影响最终的检测结果。选择合适的光学元件,并确定通道间的补偿矩阵,可以降低甚至消除荧光串扰。目前荧光补偿矩阵大多通过迭代计算获得,还没有一种简单的方法可以从混合的多通道荧光数据中找到荧光补偿矩阵。为了快速获得荧光补偿矩阵,减小计算量,采用主成分分析法(PCA)中确定主成分的方式,基于搭建的测试平台进行单一染料实验,获得染料的荧光信号在各个检测通道的分布情况,计算得到荧光补偿矩阵。通过分析补偿矩阵,发现对于搭建的硬件系统,Cy5染料对Cy5.5通道串扰较大,串扰比例为8.76%,同时Cy5.5染料对Cy5通道串扰影响也相对较大,比例约为6.2%;其次是ROX染料对HEX通道串扰,比例约为2.68%;HEX染料对FAM通道串扰,比例约为1.58%;FAM染料对HEX通道串扰相对较小,比例约为0.25%,其余通道无明显串扰,与荧光光谱反映的结果一致。采用得到的荧光补偿矩阵对单一染料实验得到的原始荧光数据进行处理,有效去除了非目标通道的荧光串扰,实现了荧光通道数据的解耦,验证了方法的可行性。最后设计了染料颜色分辨实验,将不同浓度的多种染料进行组合测试,并采用所提出的方法将得到的数据进行荧光补偿。实验结果表明,荧光通道各自的线性相关性较高,五个荧光通道的线性相关系数r均大于0.99,该结果进一步验证了该补偿方法的有效性。

Real-time fluorescence quantitative PCR is a commonly used detection method in molecular biology,mainly applied to detect DNA or RNA.However,the fluorescence data obtained by this method may feature crosstalk between fluorescence channels since there are overlapping fluorescence spectra and limitations of filter bandwidth.Such crosstalkcomplicates the PCR analysis and may ultimately affect the interpretation of detection results.Crosstalk between fluorescence channels can be reduced or eliminated by choosing appropriate filter combinations and using fluorescence crosstalk correction.Currently,the fluorescence crosstalk matrix is mostly estimated through aniterative algorithm,which is a complex method to obtain fluorescence crosstalk matrix from mixed multi-channel fluorescence data.A single dye experiment is carried out on the hardware platform to quickly calculate the fluorescence crosstalk matrix and reduce the computation.The principal component analysis(PCA)method is applied to estimate the distribution of dye fluorescence signals in each detection channel,and then the fluorescence crosstalk matrix is obtained.The crosstalk matrix shows that,for the built hardware platform,the Cy5 dye has a considerable crosstalk to the Cy5.5 channel with a crosstalk ratio of 8.76%;the Cy5.5 dye has a 6.2%crosstalk ratio to the Cy5 channel;the ROX dye has a 2.68%crosstalk ratio to the HEX channel;the crosstalk ratio of HEX dye to FAM channel is about 1.58%;the crosstalk ratio of FAM dye to HEX channelis relatively small,with only about 0.25%,and the other channels have no apparent crosstalk between each other,which is consistent with the fluorescence spectrum.The fluorescence crosstalk matrix is used to process the raw fluorescence data from the single dye experiment,which effectively removes the fluorescence data from the non-target channel and realizes the decoupling of the fluorescence channel data.The feasibility of the method is thus confirmed.Subsequently,a fluorescence separationexperiment is designed by randomly mixing various dyes of different concentrations to evaluate the quality of the crosstalk matrix s fluorescence correction.The experimental data are subject to fluorescence correction,and the linearity of the fluorescence for each dye is analyzed.The result demonstrates that the linear correlation of each fluorescent channel is high,and each linear correlation coefficient r of the five fluorescence channels exceeds 0.99,further validating the method s effectiveness.