Note on the PCR threshold standard curve

Abstract

Abstract Objective The PCR threshold standard curve is based on an exponential model of the initial phase of a PCR run where template replication efficiency is constant cycle to cycle. As such it requires that a threshold is at a level of amplified template not higher than where replication efficiency falls from its initial value. A second requirement is that all amplification profiles, both calibration and test, have the same initial efficiency. However, whether these requirements are met may not be checked, and there seems an apparent awareness that thresholds can be set higher than where efficiency has dropped from the initial value without compromising result validity. The objective of this study is to reconcile using the method without satisfying the requirement that amplification is exponential at threshold level. Results Substituting the more general requirement that profile shapes be congruent to threshold level, except for translation along the cycle axis, and a derivation of the standard curve that includes cycles beyond the exponential phase accomplishes the objective without affecting usage of the method or any prior results and enables a practicable way to verify that the second requirement for same initial efficiency is satisfied.

Keywords

• PCR
• qPCR
• Standard curve
• Threshold
• Efficiency