Bar Tzachi, Ståhlberg Anders, Muszta Anders, Kubista Mikael
Department of Chemistry and Bioscience, Chalmers University of Technology, Medicinargatan 7B, 405 30 Gothenburg, Sweden.
Nucleic Acids Res. 2003 Sep 1;31(17):e105. doi: 10.1093/nar/gng106.
Real-time PCR is becoming the method of choice for precise quantification of minute amounts of nucleic acids. For proper comparison of samples, almost all quantification methods assume similar PCR efficiencies in the exponential phase of the reaction. However, inhibition of PCR is common when working with biological samples and may invalidate the assumed similarity of PCR efficiencies. Here we present a statistical method, Kinetic Outlier Detection (KOD), to detect samples with dissimilar efficiencies. KOD is based on a comparison of PCR efficiency, estimated from the amplification curve of a test sample, with the mean PCR efficiency of samples in a training set. KOD is demonstrated and validated on samples with the same initial number of template molecules, where PCR is inhibited to various degrees by elevated concentrations of dNTP; and in detection of cDNA samples with an aberrant ratio of two genes. Translating the dissimilarity in efficiency to quantity, KOD identifies outliers that differ by 1.3-1.9-fold in their quantity from normal samples with a P-value of 0.05. This precision is higher than the minimal 2-fold difference in number of DNA molecules that real-time PCR usually aims to detect. Thus, KOD may be a useful tool for outlier detection in real-time PCR.
实时聚合酶链反应（Real-time PCR）正成为精确量化微量核酸的首选方法。为了对样本进行恰当比较，几乎所有量化方法都假定反应指数期的聚合酶链反应效率相似。然而，处理生物样本时聚合酶链反应受到抑制的情况很常见，这可能会使假定的聚合酶链反应效率相似性无效。在此，我们提出一种统计方法——动力学异常值检测（Kinetic Outlier Detection，KOD），用于检测效率不同的样本。KOD基于对测试样本扩增曲线估算的聚合酶链反应效率与训练集中样本的平均聚合酶链反应效率进行比较。在具有相同初始模板分子数的样本上对KOD进行了演示和验证，在这些样本中，dNTP浓度升高会不同程度地抑制聚合酶链反应；以及在检测两个基因比例异常的cDNA样本时。将效率差异转化为数量差异后，KOD可识别出与正常样本数量相差1.3至1.9倍且P值为0.05的异常值。这种精度高于实时聚合酶链反应通常旨在检测的DNA分子数量至少2倍的差异。因此，KOD可能是实时聚合酶链反应中异常值检测的有用工具。
