In real-time quantitative polymerase chain reaction (PCR), the standard curve between threshold cycle and logarithm of template concentration is currently the gold standard for template quantification. The efficacy of this approach is limited by the necessary assumption that all samples are amplified with the same efficiency. To overcome this limitation, a new method has been proposed in this contribution for quantitative PCR with internal standard. Unlike existing methods based upon analysis of amplification profile position, the new method tries to determine the initial quantity of the target template in a sample from the fluorescence spectrum measured at a certain point during its PCR reaction. There is no unrealistic prerequisite (e.g., constant amplification efficiency) for the successful application of the new method. The performance of the new method was evaluated by the quantification of KRAS gene in HepG2 samples. Quantitative results with recovery rates in the range of 91.2–118% were achieved by the new method. It is reasonable to expect that the new method would have a place in real-time quantitative PCR, thanks to its features of no unrealistic prerequisite, sound theoretical basis, good performance, and implementation simplicity.

在实时定量聚合酶链反应（PCR）中，阈值循环和模板浓度的对数之间的标准曲线目前是模板定量的金标准。该方法的有效性受到所有样品均以相同效率扩增的必要假设的限制。为了克服这一局限性，已提出了一种新的方法，可用于内标定量PCR。与基于扩增谱图位置分析的现有方法不同，该新方法尝试根据在PCR反应过程中某一点测得的荧光光谱确定样品中目标模板的初始量。成功应用新方法没有不现实的前提（例如恒定的放大效率）。通过对HepG2样品中的KRAS基因进行定量，评估了新方法的性能。通过新方法，定量回收率达到91.2–118％。可以预期，由于该新方法具有不切实际的先决条件，合理的理论基础，良好的性能以及实现的简便性，因此有望在实时定量PCR中占有一席之地。