Robust and Precise Quantitative Real-Time Polymerase Chain Reaction with an Amplification Efficiency-Aware Reaction Kinetics Model.

ABSTRACT

Quantitative real-time PCR (qPCR) is a method extensively used in nucleic acid testing for plants and animals. During the coronavirus disease 2019 (COVID-19) pandemic, high-precision qPCR analysis was urgently needed since quantitative results obtained from conventional qPCR methods were not accurate and precise, causing misdiagnoses and high rates of false-negative. To achieve more accurate results, we propose a new qPCR data analysis method with an amplification efficiency-aware reaction kinetics model (AERKM). Our reaction kinetics model (RKM) mathematically describes the tendency of the amplification efficiency during the whole qPCR process inferred by biochemical reaction dynamics. Amplification efficiency (AE) was introduced to rectify the fitted data so as to match the real reaction process for individual tests, thus reducing errors. The 5-point 10-fold gradient qPCR tests of 63 genes have been verified. The results of a 0.9% slope bias and an 8.2% ratio bias using AERKM exceed 4.1 and 39.4%, respectively, of the best performance of existing models, which demonstrates higher precision, less fluctuation, and better robustness among different nucleic acids. AERKM also provides a better understanding of the real qPCR process and gives insights into the detection, treatment, and prevention of severe diseases.