Dipartimento DiSUAN, Sezione di Biomatematica, Università degli Studi di Urbino Carlo Bo, Campus Scientifico Sogesta; Località Crocicchia - 61029 Urbino, Italy.
BMC Bioinformatics. 2010 Apr 12;11:186. doi: 10.1186/1471-2105-11-186.
Real-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD.
Richards function was fitted on fluorescence readings to parameterize the amplification curves. There was not a significant correlation between calculated amplification parameters (plateau, slope and y-coordinate of the inflection point) and the Log of input DNA demonstrating that this approach can be used to achieve a "fingerprint" for each amplification curve. To identify the outlier runs, the calculated parameters of each unknown sample were compared to those of the standard samples. When a significant underestimation of starting DNA molecules was found, due to the presence of biological inhibitors such as tannic acid, IgG or quercitin, SOD efficiently marked these amplification profiles as outliers. SOD was subsequently compared with KOD, the current approach based on PCR efficiency estimation. The data obtained showed that SOD was more sensitive than KOD, whereas SOD and KOD were equally specific.
Our results demonstrated, for the first time, that outlier detection can be based on amplification shape instead of PCR efficiency. SOD represents an improvement in real-time PCR analysis because it decreases the variance of data thus increasing the reliability of quantification.
实时 PCR 最近已成为绝对和相对核酸定量的首选技术。实时 PCR 中黄金标准的定量方法假设比较的样本具有相似的 PCR 效率。然而，生物样本中存在的许多因素会影响 PCR 动力学，从而干扰定量分析。在这项工作中，我们提出了一种新的策略来检测异常值样本，称为 SOD。
使用 Richards 函数拟合荧光读数以参数化扩增曲线。计算出的扩增参数（平台期、斜率和拐点的 y 坐标）与输入 DNA 的对数之间没有显著相关性，这表明这种方法可用于为每个扩增曲线生成“指纹”。为了识别异常值运行，将每个未知样本的计算参数与标准样本的参数进行比较。当由于存在生物抑制剂（如鞣酸、IgG 或槲皮素）而发现起始 DNA 分子的显著低估时，SOD 会有效地将这些扩增谱标记为异常值。随后将 SOD 与 KOD 进行比较，KOD 是目前基于 PCR 效率估计的方法。所得数据表明，SOD 比 KOD 更敏感，而 SOD 和 KOD 的特异性相同。
我们的结果首次表明，可以基于扩增形状而不是 PCR 效率来检测异常值。SOD 代表了实时 PCR 分析的改进，因为它降低了数据的方差，从而提高了定量的可靠性。
