Bias in the Cq value observed with hydrolysis probe based quantitative PCR can be corrected with the estimated PCR efficiency value

Abstract

For real-time monitoring of PCR amplification of DNA, quantitative PCR (qPCR) assays use various fluorescent reporters. DNA binding molecules and hybridization reporters (primers and probes) only fluoresce when bound to DNA and result in the non-cumulative increase in observed fluorescence. Hydrolysis reporters (TaqMan® probes and QZyme™ primers) become fluorescent during DNA elongation and the released fluorophore remains fluorescent during further cycles; this results in a cumulative increase in observed fluorescence. Although the quantification threshold is reached at a lower number of cycles when fluorescence accumulates, in qPCR analysis no distinction is made between the two types of data sets. Mathematical modeling shows that ignoring the cumulative nature of the data leaves the estimated PCR efficiency practically unaffected but will lead to at least 1 cycle underestimation of the quantification cycle (Cq value), corresponding to a 2-fold overestimation of target quantity. The effect on the target-reference ratio depends on the PCR efficiency of the target and reference amplicons. The leftward shift of the Cq value is dependent on the PCR efficiency and with sufficiently large Cq values, this shift is constant. This allows the Cq to be corrected and unbiased target quantities to be obtained.