Standardized determination of real-time PCR efficiency from a single reaction set-up

TLDR

The study proposes a computational method to estimate real‑time PCR amplification efficiency. The method uses statistical delimitation of the exponential phase, excludes non‑exponential and plateau phases with separate algorithms, and was validated on multiple targets using LightCycler data. The method achieves higher accuracy than serial‑dilution approaches, is sensitive to target concentration differences, and eliminates researcher subjectivity, enabling full automation.

Abstract

We propose a computing method for the estimation of real-time PCR amplification efficiency. It is based on a statistic delimitation of the beginning of exponentially behaving observations in real-time PCR kinetics. PCR ground fluorescence phase, non-exponential and plateau phase were excluded from the calculation process by separate mathematical algorithms. We validated the method on experimental data on multiple targets obtained on the LightCycler platform. The developed method yields results of higher accuracy than the currently used method of serial dilutions for amplification efficiency estimation. The single reaction set-up estimation is sensitive to differences in starting concentrations of the target sequence in samples. Furthermore, it resists the subjective influence of researchers, and the estimation can therefore be fully instrumentalized.