Prolonged Linear Amplification of qPCR for the Correction of Amplification Variation and the Absolute Quantification without Standard Curves

The variable amplification efficiency of each thermal cycle of qPCR obeys the Poisson distribution, and the qPCR system dynamically changes, so there must be a detection error in its quantitative analysis. Here, more than 20 cycles of the linear amplification of qPCR can be produced as the BSA hydrogel is introduced to achieve the controlled release of Taq DNA polymerase. There is a significant negative correlation between the slope of linear amplification and Ct values (r = -0.9455), and it is well evident that the slope can reflect the amplification efficiency and a linear positive correlation exists between them. Through the change in the concentration of primers in the qPCR system, an exponential equation between Ct values and the slopes can be fitted (R2 = 0.9995). The slopes and Ct values of each qPCR system can be corrected by using this equation to guarantee that there will be significant consistency in their amplification efficiency because the degree of linear fitting (R2) between Ct values and the logarithm of their corresponding concentration of the DNA template increased significantly. By this time, the accurate amplification efficiency can be calculated in a known multiple of two initial concentrations of DNA templates. With the aid of the relationship between the known primer concentration and the fluorescence intensity at the end of PCR (End RFU), the initial concentrations of DNA templates can be reversely calculated in the absence of standard curves.