Variation in PCR Efficiencies between Quantification Standards and Clinical Specimens using Different Real-Time Quantitative PCR Interpretation Methods

Abstract

The analysis of the Ct and standard curve produced by real-time polymerase chain reaction (PCR) is a well-established method for the quantification of nucleic acids. However, this method assumes that the PCR efficiency between the unknown specimen and standard is equal, resulting in the possibility of significant inaccuracies due to the presence of inhibitory agents in the unknown specimen. Although numerous methods have been proposed to correct this issue, the understanding of the differences in PCR efficiencies in clinical samples is limited. In this study, 1185 cytomegalovirus (CMV) DNA real-time PCR test results from 106 batches were analyzed. The PCR efficiencies were calculated using the cpD2, maxE, Cy0, maxRatio and window-of-linearity (WoL) methods. The concentrations were calculated using the cpD2, Cy0, maxRatio, WoL, and take off point (TOP) methods. The coefficient of variation (CV) in the efficiency of the quantification standards was less than 5% in all methods. Positive samples with high quantification values demonstrated lower PCR efficiency compared to the quantification standards. This suggests possible inaccuracies in quantification using quantification standards in clinical samples.