Comparison of temperature dynamics of various thermal cycler calibration methods

Abstract

A multitude of testing laboratories, including medical, forensic, food, water and environmental testing laboratories, use thermal cycler equipment for Polymerase Chain Reaction (PCR) based DNA testing. PCR reactions are temperature critical and therefore these thermal cyclers require regular calibration to ensure correct functioning. Over time, a variety of calibration and verification methods have been published. Most of these methods are not traceable to the International Systems of Units (SI) and are connected to substantial measurement uncertainties, and therefore inapt for application in metrology. Physical temperature sensor based methods have found to be the most appropriate methods. These temperature sensor based methods can be categorized in in-tube and in-well methods. The in-tube methods measure temperature inside the PCR reaction tubes, which are placed in the wells of the thermal cycler heating block. The in-well methods measure temperature directly in the wells of the thermal cycler heating block. Both in-tube and in-well methods can be designed to be metrological traceable. However, the associated measurement uncertainty can vary substantially between in-tube and in-well methods. This paper compares the temperature dynamics of in-tube and in-well methods and discusses systematic and random errors which can contribute to the measurement uncertainty of both methods.