12.2.3. qPCR dynamic range and quantitation limit

PCR is an exponential (i.e. logarithmic) amplification process that is extremely consistent (i.e. predictable) over the entire reaction (35~40 cycles) and over a large range of initial target concentrations (at least 106-fold). This dynamic range and the quantitation limits of qPCR are determined using a 10-fold serial dilution series of known concentrations of (cloned) target DNA. A standard calibration curve is generated by linear regression of the quantification cycle (Cq) at which the PCR product is detected vs. the log10[target copy number]. The resulting algebraic equation:

Cq = a * log10[target] + b               (where ‘a’ is the slope and ‘b’ the intercept)

is then used to estimate the amount of target in a sample, given the Cq value (Bustin et al., 2009). For accurate calibration of the curve and determining the error associated with data conversion, at least three independent trials of three independent 10-fold serial dilutions should be run. For each series and trial, the known amounts of target in each dilution are compared to the theoretical amounts estimated from the calibration curve, to obtain the individual bias, which is the averaged for all series and trials to obtain the mean bias (mb) at each dilution (an example is shown in Table 7). These values are then used to calculate the standard deviation of the obtained values (SD), and the uncertainty of the linearity is obtained by the formula ULINi = 2[√ SD² + mb²]. The combined linearity uncertainly is defined for the entire calibration range by the formula ULIN = │√ ΣULINi2 / k│ where k is the number of dilution levels. The quantitation limit of the assay is then set at the target concentration of the calibration range.